Treating red blood cell solutions with anti-viral agents

ABSTRACT

Methods and compositions for treating pathogens in material are described, including methods of decontaminating human fluids prior to processing in the clinical laboratory and methods for decontaminating blood products prior to in vivo use. The techniques handle large volumes of human serum without impairing the testing results.

This Application is a continuation of Application Ser. No. 08/465,545filed Jun. 5, 1995 now abandoned.

FIELD OF THE INVENTION

The invention generally relates to new compounds and methods for the invitro inactivation of pathogens in biological material intended for invitro or in vivo use, and in particular the inactivation of pathogens insolutions containing red blood cells, prior to clinical testing ortransfusion.

BACKGROUND

The presence of pathogens in blood products, as well as other biologicalmaterials, is recognized as a significant health problem to healthworkers as well as recipients of the materials.

With regard to health workers, a great volume of human fluids is handleddaily as part of the routine monitoring of hospital patients byobtaining and testing human fluids (blood, urine, spinal fluid, etc.).Typically, each admitted patient has at least a tube of blood collectedevery day by a phlebotomist. During the transferring, portioning andtesting process, each sample tube is handled by a clinical worker whileits contents are exposed. This intensive handling of potentiallyinfectious human fluids is not without health risk. The OccupationalSafety and Health Administration (OSHA) estimates that over five millionhealth workers, including hospital laboratory workers, are exposed toblood borne-pathogen infections in the work place annually. The pathogenresponsible for the overwhelming majority of infections is the hepatitisB virus (HBV). The Center for Disease Control (CDC) estimates there aretwelve thousand cases of HBV infection among health workers each year.Of these cases, over five hundred require hospitalization andapproximately two hundred and fifty of these patients die (i.e. fromfulminant hepatitis, cirrhosis or liver cancer). See Guidelines forPrevention of Transmission of HIV and HBV to Health-Care and PublicSafety Workers, CDC (February 1989). Most full time laboratory employeescontract hepatitis at least once during their career. Indeed, up to onethird of all health care workers show serological evidence of a previousHBV infection. Id.

Following the recognition of Acquired Immunodeficiency Syndrome (AIDS),clinical laboratories have instituted additional precautions. Forexample, rather than using manually positioned plastic inserts tomaintain the separation of cells from serum after samples arecentrifuged, a “gel” is now available that is in the empty tube at thetime the blood is drawn. When the tube is centrifuged the cells go belowthe gel while the serum remains above. While the separation can bemaintained in this manner without as much sample handling, this does notreduce the handling of the technologist at the point of analysis.Unfortunately, infectious virus can persist in a liquid or dried statefor prolonged periods of time, possibly even at elevated temperatures.Resnick et al., JAMA 255:1887 (1986).

Preventative measures such as gloves and eye-wear are not completesolutions to the problem. Accidents in the laboratory or clinictypically involve exposure over a larger portion of the body and diseasecan be transmitted through the skin and mucous membranes. Morbidity andMortality Weekly Report 36:285 (1987).

Clearly, there remains a need for a more adequate solution to bloodborne-pathogen infections in the work place. Such a solution shouldserve as a protection against a wide range of pathogens. Furthermore,the mechanics of the solution should not unduly interfere withoperations of a laboratory or blood bank.

Another significant problem is the contamination of the blood supply forin vivo use. The safety of the blood supply continues to be threatenedby the transmission of pathogens by transfusion. While the threat posedby the human immunodeficiency virus (HIV) and the Acquired ImmuneDeficiency Syndrome (AIDS) is now widely publicized, contamination ofblood products with a number of other blood-borne infectious viralagents is of even greater concern. See R. Y. Dodd, In: TransfusionMedicine in the 1990's (American Assoc. Blood Banks 1990) (S. J. Nance,ed.). For example, in the United States, it is estimated that up to ten(10) percent of multiply transfused recipients develop hepatitisaccounting for many thousands of cases annually.

Whole blood collected from volunteer donors for transfusion recipientsis typically separated into its components: red blood cells, platelets,and plasma. Each of these fractions are individually stored and used totreat a multiplicity of specific conditions and disease states.

The red blood cell component is used primarily to treat trauma, chronicanemia, and blood loss due to surgery (particularly cardiac and liversurgery), including postoperative bleeding. D. M. Surgenor et al.Transfusion 32:458 (1992). Approximately twelve (12) million units ofred cells are transfused into approximately four (4) million recipientsannually in the United States alone. E. L. Wallace et al. Transfusion33:139 (1993).

The safety of the blood supply cannot be assured by merely testing theblood for pathogens before transfusion. Most testing relies on thedetection of antibodies to the pathogen in the prospective blood donor.It is now well-documented that infectious agents can be transmitted by“seronegative” blood donors, i.e. donors that have no detectableantibodies to the pathogen. For example, thirteen cases oftransfusion-related AIDS have been reported to the Centers for DiseaseControl (CDC) among recipients of blood that was pre-tested and foundnegative for antibody to the HIV-1 virus.

Clerical errors and other mistakes further expose patients tocontaminated, incorrectly tested or mislabeled blood. To complicate theproblem, one bad unit can create several victims, since whole blood isroutinely split into components. Mistakes are not infrequent in bloodbanks. Since the beginning of 1990, 29,586 blood bank errors andaccidents have been reported to the FDA. “How Safe Is Our Blood,” U.S.News and World Report, Jun. 27, 1994, 68-78. Recalls by blood centers ofblood released in error are generally ineffective because they takeplace months or years after the blood products have been transfused.

An alternative approach to eliminate transmission of diseases throughblood products is to develop a means to inactivate pathogens-intransfusion products. Some of these techniques such as heat [J.Hilfenhous et al. J. Biol. Std. 70:589 (1987)], solvent/detergenttreatment [B. Horowitz et al. Transfusion 25:516 (1985)],gama-irradiation [G. Moroff et al. Transfusion 26:453 (1986)] or UValone [K. N. Proudouz et al. Blood 70:589 (1987)] are completelyincompatible with maintenance of red cell function.

Another means to inactivate pathogens is the use of methylene blue. S.J. Wagner et al. examined methylene blue as a virucidal for red cellsolutions. S. J. Wagner et al. Transfusion 33:30 (1993). Photo treatmentof red cells with methylene blue was found to cause loss of ATP,enhanced ion permeability, and binding of autologous immunoglobulin(IgG) to the red cell surface. It was speculated that some general (andundesirable) modification of the red cell membrane occurs as a result ofthe treatment.

Yet another approach is to deplete the red cell product of contaminatinglymphocytes which may harbor viral pathogens. Both leukodepletion withfilters and freeze/thaw procedures have been examined. S. M. Bruisten etal. Transfusion 30:833 (1990). Complete removal of lymphocytes, however,cannot be achieved with such methods. Furthermore, leukodepletion doesnot address cell-free virus. Thus, this approach is not sufficient torender blood completely safe.

Finally, there is the approach of avoiding blood and using bloodsubstitutes. Hemoglobin solutions, perfluorocarbon emulsions andvesicle-encapsulated hemoglobin have all been suggested as candidates.Unfortunately, each of these has been shown to be inadequate as ageneral substitute. See R. M. Winslow In: Blood Safety: CurrentChallenges (S. J. Nance ed.) (AABB 1992) (pp. 151-167).

In sum, there is a need for a means of inactivating viral pathogens inred blood cell solutions. This approach must be effective withoutcausing harm to the blood product or the transfusion recipient.

SUMMARY OF THE INVENTION

The present invention generally relates to new compounds and methods forthe in vitro inactivation of pathogens in biological material intendedfor in vitro or in vivo use, and in particular the inactivation ofpathogens in solutions containing red blood cells, prior to clinicaltesting or transfusion. In accordance with the present invention, acompound having a nucleic acid binding ligand and a mustard group isselectively employed to treat contamination by nucleic acid-containingmicroorganisms, including pathogenic viruses. Without intending tosuggest a mechanism for the present invention, such compounds arealkylating agents.

The present invention contemplates a method of decontaminating pathogensin a biological composition, comprising: adding a compound having anucleic acid binding ligand and a mustard group to a biologicalcomposition suspected of containing pathogens, to create a mixture, saidcompound reaching a final concentration sufficient to inactivatesubstantially all of said pathogens, and incubating said mixture withoutsignificant damage to said biological composition. In one embodiment,the compound is added to the biological composition to a finalconcentration of said compound of between 1 μg/ml and 250 μg/ml. Inanother embodiment, the mixture is incubated for between 1 minute and 48hours. In an embodiment of the present invention, the compound is addedto the biological composition, the compound is in a mixture comprisingdextrose, sodium chloride, mannitol, adenine and H₂O.

The present invention contemplates that the biological compositioncomprises a blood product. The present invention also contemplates anadditional step : c) transfusing said blood product into a mammal. Inone embodiment, the blood product transfused comprises red blood cells,or specifically, red blood cell concentrate.

The present invention contemplates the inactivation of both viral andbacterial pathogens. One compound contemplated by the present inventionfor this method is N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediamine.However, the invention contemplates that more than one of said compoundsmay be added to the biological composition. Further, the inventioncontemplates a step c): after incubating said mixture, removing saidcompound from said biological composition with an adsorbent material.

Specifically, the present invention contemplates a method ofinactivating pathogens in a blood product, comprising: a) adding acompound having a nucleic acid binding ligand and a mustard group to ablood product comprising red blood cells suspected of containingpathogens, to create a mixture, said compound reaching a finalconcentration sufficient to inactivate substantially all of saidpathogens, and b) incubating said mixture for between 1 minute and 48hours, without significant damage to said red blood cells. The compoundmay be added to the blood product to a final concentration of a compoundhaving a nucleic acid binding ligand and a mustard group of between 1μg/ml and 250 μg/ml. As an additional component, the inventioncontemplates that when the compound having a nucleic acid binding ligandand a mustard group is added to the blood product, the compound is in amixture comprising dextrose, sodium chloride, mannitol, adenine and H₂O.In another embodiment, the method further comprises: c) transfusing saidblood product into a mammal. The present invention contemplates theinactivation by this method of both viral and bacterial pathogens.Further contemplated is a blood product decontaminated by this method.Various compounds are contemplated for use in this method, including:N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediamine.Further, more than one of said compounds may be added to said bloodproduct. The present invention contemplates the additional stepcomprising: c) after incubating said mixture, removing said compoundfrom said biological composition with an adsorbent material.

In yet another embodiment, the present invention contemplates a methodof inactivating pathogens in a clinical sample intended for in-vitroclinical testing, comprising, in the following order: a) providing, inany order, i) N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediamine,and ii) a clinical sample intended for in vitro clinical testingsuspected of being contaminated with pathogens; b) adding a compoundhaving a nucleic acid binding ligand and a mustard group to saidclinical sample, to create a mixture, c) incubating said mixture forbetween 1 minute and 48 hours, and d) measuring the level of a clinicalchemistry analyte in said clinical sample. One compound that iscontemplated in this method is N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediamine,which is preferably added to reach a concentration of N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4pentanediaminesufficient to inactivate substantially all of said pathogens withoutsignificant damage to said clinical sample. Additionally, when N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediamineis added to said clinical sample, N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediaminemay be in a mixture comprising dextrose, sodium chloride, mannitol,adenine and H₂O.

The present invention specifically contemplates that said clinicalsample comprises red blood cells, and that the pathogens inactivated maycomprise either bacterial pathogens or viral pathogens. In oneembodiment, measuring the level of a clinical chemistry analyte in saidclinical sample is performed without significant damage to said clinicalsample.

In yet another embodiment, the present invention contemplates acomposition of matter, comprising:5-[N,N-bis(2-chloroethyl)amino]methyl-8-methoxypsoralen.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing reduction in titer of R17 treated with varyingconcentrations of quinacrine mustard in either Adsol or dimethylsulphoxide (DMSO). The horizontal dotted line represents the limit ofdetection of the assay used.

FIG. 2 is a graph showing inactivation of R17 by quinacrine mustard as afunction of hematocrit.

FIG. 3 is a graph showing the inactivation kinetics of quinacrinemustard.

FIG. 4 is a graph showing the reduction in titer of R17 as a function oftime of incubation of quinacrine mustard in Adsol.

FIG. 5 is a graph showing the reduction in R17 inactivation activity asa function of time when incubated in the presence of either Adsol, redblood cells or Amberlite XAD-16™.

FIG. 6 is a graph showing the effects of quinacrine mustard at varyingconcentrations on extra-cellular potassium levels.

FIG. 7 is a graph showing the reduction in titer of R17 treated withvarying concentrations of quinacrine mustard; the horizontal dotted linerepresents the limit of detection of the assay used.

FIG. 8 is a graph showing the activity of quinacrine mustard, afterincubation in red blood cells, with or without the presence of AmberliteXAD-16™, in an Ames assay using strain TA 1537.

FIG. 9 is a graph showing the inactivation of a bacterial strain,Staphylococcus Epidermis, using quinacrine mustard at varyingconcentrations.

DESCRIPTION OF THE INVENTION

The present invention generally relates to new compounds and methods forthe in vitro inactivation of pathogens in biological material intendedfor in vitro or in vivo use, and in particular the inactivation ofpathogens in solutions containing red blood cells, prior to clinicaltesting or transfusion. In accordance with the present invention, acompound having a nucleic acid binding ligand and a mustard group isselectively employed to treat contamination by nucleic acid-containingmicroorganisms, including pathogenic viruses and bacteria.

I. COMPOUNDS OF THE PRESENT INVENTION

Red blood cell decontamination methods using photoactivated compoundshave in the past encountered a problem due to the absorbency, byhemoglobin, of light at wavelengths necessary to activate compounds.Thus, even though the previous methods would inactivate pathogens inother media, they are inefficient in the presence of red blood cells. Incontrast, the present invention contemplates a method of sterilizationcapable of effectively inactivating pathogens even in red cellconcentrates [hematocrits ranging from 1% to 60% or higher].

The present invention contemplates treating red blood cell solutionswith a compound which inactivates pathogens without requiring exposureto light. The advantage of the present invention for inactivation influids for transfusion is two-fold. First, light is not required,allowing for a less complex technology for inactivation. Second, thedecontamination compound is completely reacted after a short amount oftime. The treatment is complete in several minutes or hours, dependingon the compound used. Material which does not react with nucleic acidsor another biomolecule hydrolyzes, leaving no compound to be transfused.

Without intending to be limited to any particular mechanism of action ofthe present invention, compounds of the present invention have twocharacteristics in common. The first characteristic is that they bindnucleic acid non-covalently. The second is that they have at least onemustard group.

A. Non-covalent Nucleic Acid Binding Group

A compound which binds nucleic acid has a “nucleic acid binding ligand”,herein defined as a group which has an affinity for and can bind tonucleic acids non-covalently. There are several modes of binding tonucleic acids. Compounds which bind by any of the following modes,combinations of them, or other modes are considered nucleic acid bindingligands. While the invention is not limited to the following compounds,some examples of nucleic acid binding ligands are: a) intercalators,such as acridines, acridones, proflavin, acriflavine, actinomycins,anthracyclinones, beta-rhodomycin A, daunamycin, thiaxanthenones,miracil D, anthramycin, mitomycin, echinomycin, quinomycin, triostin,diacridines, ellipticene (including dimers, trimers and analogs),norphilin A, fluorenes and flourenones, fluorenodiamines, quinacrine,benzacridines, phenazines, phenanthradines, phenothiazines,chlorpromazine, phenoxazines, benzothiazoles, xanthenes andthio-xanthenes, anthraquinones, anthrapyrazoles, benzothiopyranoindoles,3,4-benzpyrene, benzopyrene diol epoxidie, 1-pyrenyloxirane,benzanthracene-5,6-oxide, benzodipyrones, benzothiazoles, quinolones,chloroquine, quinine, phenylquinoline carboxamides, furocoumarins, suchas psoralens and isopsoralens, ethidium salts, propidium, coralyne,ellipticine cation and derivatives, polycyclic hydrocarbons and theiroxirane derivatives, and echinimycin; b) minor groove binders such asdistamycin, mitomycin, netropsin, other lexitropsins, Hoechst 33258 andother Hoechst dyes, DAPI (4′,6′-diamidine-2-phenylindole), berenil, andtriarylmethane dyes; c) major groove binders such as aflatoxins; d)molecules that bind by electrostatics (phosphate backbone binders), suchas spermine, spermidine, and other polyamines; e) nucleic acids oranalogues which bind by such sequence specific interactions as triplehelix formation, D-loop formation, and direct base pairing to singlestranded targets.

While not limited to any particular mechanism, it is believed that thenucleic acid binding ligand functions as a carrier (or anchor) thattargets (or directs) the molecule to nucleic acid, interactingnon-covalently therewith.

B. Mustard Group

The second characteristic that compounds of the present invention havein common is that they contain at least one mustard group. A “mustardgroup” is defined here as including mono or bis haloethylamine groups,and mono haloethylsulfide groups.

The present invention is not limited strictly to mustards. It isbelieved that mustards can form reactive intermediates such asaziridinium or aziridine complexes and sulfur analogs of thesecomplexes. The present invention also contemplates functional groupsthat are the equivalent of mustards, such as epoxides.

While not limited to any particular mechanism, compounds having mustardgroups are known to react with nucleic acids to form covalent complexeswhich inhibit nucleic acid replication. They are typically solids that,upon dissolution in a medium which contains nucleophiles, completelyreact within minutes or hours. Some examples are shown below.

Nitrogen mustards are thoroughly described in the literature. E.g., seeGravatt, G. L., et al., “DNA-Directed Alkylating Agents. 4.4-Anilinoquinoline-Based Minor Groove Directed Aniline Mustards,” J.Med. Chem. 34:1552 (1991); Cummings, J., et al., “Determination ofReactive Nitrogen Mustard Anticancer Drugs in Plasma by High-PerformanceLiquid Chromatography Using Derivatization,” Anal. Chem. 63:1514 (1991).They are known to be potent alkylators of nucleic acid and due to thismode of action, they have been widely studied as anti-tumor agents.Several have found practical use in the clinic (e.g. aniline mustard,chlorambucil, melphalan).

One class of nitrogen mustards is the aniline mustards. These compoundshave at least one haloethylaminoaniline group on them, where thehaloethyl may be mono or bis. An example of a bis(haloethyl)aminoanilinegroup appears below (where R is the point of linkage to other groups):

A specific aniline mustard group is the acridine carried anilinemustards (described in Gravatt, et al., J. Med. Chem. 34:1552), where Rcomprises a linking group (for example O, CH₂, S, COHN, or CO, however,other linking groups are contemplated) which links the mustard group toa second component, an acridine group. An example of the components of a9-aminoacridine carried aniline mustard appears below (where X is thelinking group):

The present invention demonstrates that a specific compound having anucleic acid binding ligand and a mustard group, N1,N1-bis(2-chloroethyl)-N4-(6-chloro-2-methoxy-9-acridinyl)-1,4-pentanediaminedihydrochloride (“quinacrine mustard”), is useful as an antiviral agentfor red cells. Quinacrine mustard is commercially available (fromAldrich, Milwaukee, Wis., as quinacrine mustard dihydrochloride hydrate,structure shown below).

II. MATERIALS FOR DECONTAMINATION

The present invention contemplates novel compounds and a new use forcompounds having a nucleic acid binding ligand and a mustard group: theinactivation of viruses and bacteria in blood, blood products and otherbiological compositions. While not an exclusive list, the followingbiological compositions are contemplated, and are referred to generallyas “samples”. Of the blood and blood components contemplated, exemplarycompositions include whole blood, packed red cells, platelets, plasma(fresh or fresh frozen plasma), and proteins derived from blood or bloodcomponents. Blood components also encompass plasma protein portion,antihemophilic factor (AHF, Factor VIII); Factor IX and Factor IXcomplex (Factors II, VII, IX and X); fibrinogens, Factor XIII,prothrombin and thrombin (Factor II and IIa); immunoglobulins (e.g. IgA,IgD, IgE, IgG and IgM and fragments thereof e.g. Fab, F(ab′)₂, and Fc);hyper-immune globulins as used against tetanus and hepatitis B;cryoprecipitate; albumin; interferons; lymphokines; and transferfactors. The present invention also contemplates, as part of blood andblood products, a synthetic version of any blood or blood product.

Other biological compositions which are contemplated by the presentinvention include vaccines, recombinant DNA produced proteins,oligopeptide ligands, etc. Biological compositions also encompassclinical samples other than blood and blood components, such as urine,sputum, feces, spinal fluid, and other materials removed from mammalsfor clinical testing.

III. INACTIVATION OF PATHOGENS

The present invention contemplates treating a blood product with acompound having a nucleic acid binding ligand and a mustard group toinactivate contaminating pathogen nucleic acid sequences before usingthe blood product.

A. Inactivation In General

The term “inactivation” is here defined as the altering of the nucleicacid of a unit of pathogen so as to render the unit of pathogenincapable of replication. This is distinct from “total inactivation”,where all pathogen units present in a given sample are renderedincapable of replication, or “substantial inactivation,” where most ofthe pathogen units present are rendered incapable of replication.“Inactivation efficiency” of a compound is defined as the level ofinactivation the compound can achieve at a given concentration ofcompound or dose of irradiation. For example, if 100 μM of ahypothetical compound X inactivated 5 logs of HIV virus whereas underthe same experimental conditions, the same concentration of compound Yinactivated only 1 log of virus, then compound X would have a better“inactivation efficiency” than compound Y.

To appreciate that an “inactivation” method may or may not achieve“total inactivation,” it is useful to consider a specific examples Abacterial culture is said to be inactivated if an aliquot of theculture, when transferred to a fresh culture plate and permitted togrow, is undetectable after a certain time period. A minimal number ofviable bacteria must be applied to the plate for a signal to bedetectable. With the optimum detection method, this minimal number is 1bacterial cell. With a sub optimal detection method, the minimal numberof bacterial cells applied so that a signal is observed may be muchgreater than 1. The detection method determines a “threshold” belowwhich the “inactivation method” appears to be completely effective (andabove which “inactivation” is, in fact, only partially effective).

B. Inactivation of Potential Pathogens

The same considerations of detection method and threshold exist whendetermining the sensitivity limit of an inactivation method for nucleicacid. Again, “inactivation” means that a unit of pathogen is renderedincapable of replication.

In the case of inactivation methods for material to be used by humans,whether in vivo or in vitro, the detection method can theoretically betaken to be the measurement of the level of infection with a disease asa result of exposure to the material. The threshold below which theinactivation method is complete is then taken to be the level ofinactivation which is sufficient to prevent disease from occurring dueto contact with the material. It is recognized that in this practicalscenario, it is not essential that the methods of the present inventionresult in “total inactivation”. That is to say, “substantialinactivation” will be adequate as long as the viable portion isinsufficient to cause disease. Thus “substantially all” of a pathogen isinactivated when any viable portion of the pathogen which remains isinsufficient to cause disease. The inactivation method of the presentinvention renders nucleic acid in pathogens substantially inactivated.In one embodiment, the inactivation method renders pathogen nucleic acidin blood preparations substantially inactivated.

Without intending to be limited to any method by which the compounds ofthe present invention inactivate pathogens, it is believed thatinactivation results from alkylation of portions of the pathogen nucleicacid. Further, while it is not intended that the inactivation method ofthe present invention be limited by the nature of the nucleic acid; itis contemplated that the inactivation method render all forms of nucleicacid (whether DNA, mRNA, etc.) substantially inactivated.

When a compound having a nucleic acid binding ligand and a mustard groupis used to modify nucleic acid, the interaction of the pathogen nucleicacid (whether DNA, mRNA, etc.) with the compound preferably preventsreplication of the pathogen, such that, if a human is exposed to thetreated pathogen, infection will not result.

“Synthetic media” is herein defined as an aqueous synthetic blood orblood product storage media. In one embodiment, the present inventioncontemplates inactivating blood products in synthetic media comprising abuffered saline solution.

The present method inactivates nucleic acid based pathogens present inblood through a single procedure. Thus, it has the potential toeliminate bacteria, protozoa, and viruses as well. It is not intendedthat the present invention be limited by the number or nature ofpathogens inactivated. Importantly, however, the treatment of thepresent invention has been found to block the replication of the HIVvirus. Had an effective decontamination method been available prior tothe advent of the AIDS pandemic, no transfusion associated HIVtransmission would have occurred. Decontamination based on compoundshaving a nucleic acid binding ligand and a mustard group has thepotential to eliminate all infectious agents from the blood supply,regardless of the pathogen involved.

C. Selecting Compounds for Inactivation of Pathogens

In order to evaluate a compound to decide if it would be useful in thedecontamination methods of the present invention, two importantproperties should be considered: 1) the compound's ability to inactivatepathogens and 2) its mutagenicity after treatment. The ability of acompound to inactivate pathogens may be determined by several methods.One technique is to perform a bacteriophage screen; an assay whichdetermines nucleic acid binding of test compounds. A screen of thistype, an R17 screen, is described in detail in an example, below. If theR17 screen shows inactivation activity, it is useful to directly testthe compound's ability to inactivate a virus. One method of performing adirect viral inactivation screen is described in detail in an examplebelow for cell free HIV.

The R17 bacteriophage screen is believed to be predictive of HIVinactivation efficiency, as well as the efficiency of compounds againstmany other viruses. R17 was chosen because it was expected to be a verydifficult pathogen to inactivate. It is a small, single stranded RNAphage. Without intending to be limited to any means by which the presentinvention operates, it is expected that shorter pieces of nucleic acidare harder to inactivate because they provide a smaller target for thecompound. Thus it is expected that under conditions that result in theinactivation of R17 the inactivation of many viruses and bacteria willalso be obtained.

The cell free HIV screen complements the R17 screen by affirming that agiven compound which has tested positive in R17 will actually workeffectively to inactivate viruses. Thus, if a compound shows activity inthe R17 screen, it is next tested in the viral inactivation screen.

The second property that is important in testing a compound for use inmethods of the present invention is mutagenicity after treatment. Themost widely used mutagen/carcinogen screening assay is the Ames test.This assay is described by D. M. Maron and B. N. Ames in MutationResearch, 113: 173 (1983) and a specific screen is described in detailin an example, below. The Ames test utilizes several unique strains ofSalmonella typhimurium that are histidine dependent for growth and thatlack the usual DNA repair enzymes. The frequency of normal mutationsthat render the bacteria independent of histidine (i.e., the frequencyof spontaneous revertants) is low. The test allows one to evaluate theimpact of any residual chemical entities that remain after treatment onthis revertant frequency.

Because some substances are not mutagenic by themselves, but areconverted to a mutagen by metabolic action, the compound to be tested ismixed with the bacteria on agar plates along with the liver extract. Theliver extract serves to mimic metabolic action in an animal. Controlplates have only the bacteria and the extract.

The mixtures are allowed to incubate. Growth of bacteria (if any) ischecked by counting colonies. A positive Ames test is one where thenumber of colonies on the plates with mixtures containing the compoundsignificantly exceeds the number on the corresponding control plates.

When known carcinogens are screened in this manner with the Ames test,approximately ninety percent are positive. When known noncarcinogens aresimilarly tested, approximately ninety percent are negative.

A compound (X) can be evaluated as a potential decontamination compoundfor use in the present invention, as shown in Table 1, below. X isinitially evaluated in Step I. X is screened in the R17 assay, in thepresence of red blood cells, at several different concentrations between4 and 320 μM, as explained in an example below. If the compound showsinactivation activity greater than 1 log inactivation of R17 (log kill)in the R17 screen at any concentration, the compound is then screened inthe cell free HIV assay, Step II, as explained in an example below. Ifthe compound shows inactivation activity greater than 1 log inactivationof HIV (log kill) in the cell free HIV assay, the compound is a usefulagent for inactivation of pathogens in clinical test samples. If thecompound is being evaluated for decontamination of biological materialsto be used in vivo, it is then taken through Step III. A biologicalmaterial decontaminated by a method of the present invention is screenedin the Ames assay to determine whether any compound that remains afterdecontamination is mutagenic. Finally, if the residual material does notshow significant mutagenicity in the Ames assay, the compound isidentified as a useful agent for inactivation of pathogens in productsto be used in vivo as well.

TABLE 1 STEP SCREEN RESULT INTERPRETATION I R17 >1 log kill by anypotential compound, concentration go to step 2 <1 log kill compound isineffective as an inactivation treatment II Viral >1 log kill by anyuseful for clinical sample Inactivation concentration decontamination goto step 3 <1 log kill compound is ineffective as an inactivationtreatment III Ames less mutagenic useful agent for inactivation than AMT

By following these instructions, a person can determine which compoundswould be appropriate for use in methods of the present invention.

D. Delivery and Removal of Compounds for Inactivation

The present invention contemplates several different formulations androutes by which the compounds described herein can be delivered in aninactivation method, and where desired, removed. This section is merelyillustrative, and not intended to limit the invention to any form ormethod of treatment with the compounds.

The compounds of the present invention may be introduced in aninactivation method in several forms and at various times, which maydepend on the purpose for which the blood preparation is decontaminated.The compounds may, for example, be introduced as an aqueous solution inwater, saline, a synthetic media or a variety of other media. Thecompounds may alternatively be provided as dry formulations, with orwithout adjuvants. Further, the compounds may be introduced alone, or ina “cocktail” or mixture of several different compounds. In a preferredembodiment, a compound having a nucleic acid binding ligand and amustard group is employed at a concentration less than 250 μM.

The compounds can be mixed directly with the blood or blood product orprepared as a solution or suspension in a bio-compatible fluid [such asAdsol (the contents of which are set forth in the Experimental section,below) or an organic solvent (e.g. dimethyl sulfoxide (DMSO), ethanol,glycerin, polyethylene glycol (PEG) or polypropylene glycol)] and thenmixed with the blood. The new compounds may also be provided atdifferent points in the inactivation process. For example, the compoundmay be introduced to the reaction vessel, such as a blood bag, at thepoint of manufacture. Alternatively, the compound may be added to thematerial to be sterilized after the material has been placed in thereaction vessel.

1. Decontamination of Clinical Samples.

A clinical sample is defined as any material removed from mammals forclinical testing, including, but not limited to blood and bloodcomponents, urine, sputum, feces, bone marrow, and spinal fluid. A serumanalyte is defined here as a component found in clinical samples whichis measured in clinical chemistry tests. Examples of serum analytesinclude, but are not limited to: glucose, blood urea nitrogen,creatinine, blood urea nitrogen/creatinine ratio, sodium, potassium,chloride, magnesium, calcium, phosphorous inorganic, total protein,albumin, total globulin, albumin/globulin ratio, billirubin, alkalinephosphatase, lactate dehydrogenase, glutamate transferase, aspartatetransaminase, alanine aminotransferase, uric acid, iron, triglycerides,and cholesterol.

In the decontamination of clinical samples, the goal is to decontaminatethe sample so that infectious agents cannot be transferred to clinicallaboratory workers. Because the samples will not be transfused into arecipient, there is less concern that residual compound be removed fromthe sample. Thus scrubbing techniques may not be desired. The presentinvention contemplates that the compound may be in the clinical sampletest tube prior to drawing the sample from the patient, or it may beadded after drawing. Once the compound has contacted the sample, thesample preferably is thoroughly mixed, then incubated. The sample maythen be screened in the desired panel of clinical chemistry testswithout concern for spreading infectious diseases.

2. Decontamination of Blood Products for Transfusion.

The compound for decontamination may be introduced to the whole bloodprior to fractionating, by adding to the blood bag before or after bloodis drawn. Alternatively, the compound may be added after fractionationof the blood, decontaminating the individual fractions.

In products for transfusion, in some cases it may be desirable to removeresidual compound or chemical products of the reaction after treatmentof the product but prior to transfusion. The present inventioncontemplates the removal, or “scrub” of the compound from the bloodproduct post illumination and prior to transfusion. In one embodiment,any residual compound or chemical product may be removed using anadsorbent material. Examples of adsorbent materials which may be used inthe present invention include, but are not limited to: activatedcharcoal (either uncoated or coated with a polymer), silica, reversephase silica, polymeric adsorbents, and modified polymeric adsorbents.The present invention contemplates several ways for the introduction ofthe adsorbent material to the blood products for transfusion. Theadsorbent may be mixed directly with the blood products and subsequentlyfiltered out. Alternatively, the blood products could be passed througha filter containing the adsorbent material.

3. Decontamination of Vaccines and Other Biological Compositions

Vaccines and other biological compositions which are not derived fromblood, such as recombinant DNA produced proteins and oligopeptideligands, may also be decontaminated using methods of the presentinvention. Recombinant DNA produced proteins often are manufactured inlarge quantities in host organisms. Introduction of the decontaminationcompound may occur prior to amplification, so that as the host organismsgrow, the compound is incorporated into the organism. Alternatively, thecompound may be added after manufacture, but before the product isintroduced into a mammal.

Removal of the compound before use may be desired here as well as withblood products for transfusion. Those methods mentioned above applyequally well in the case of vaccines and other biological compositions.

V. PRESERVATION OF BIOCHEMICAL PROPERTIES OF TREATED MATERIAL

When treating blood products to be used in vivo, one must ask whetherthe process or the compounds used alter the in vivo activity of thetreated material. For example, red blood cell transfusion is a wellestablished efficacious treatment for patients suffering large bloodloss. However, if the inactivation treatment used greatly reduces the invivo life of the red blood cells, then the treatment has no practicalvalue. The compounds of the present invention are useful in inactivationprocedures because the reaction can be carried out at temperaturescompatible with retaining biochemical properties of blood and bloodproducts. But not all methods of pathogen inactivation will inactivatewithout significantly lowering the biological activity of thedecontaminated material. Previously known compounds and protocols forinactivation have necessitated both exposure to light and the subsequentremoval of molecular oxygen from the reaction before and during theexposure, to prevent damage to blood products from oxygen radicalsproduced during irradiation. See L. Lin et al., Blood 74:517 (1989);U.S. Pat. No. 4,727,027, to Wiesehahn. The present invention may be usedto decontaminate blood products without light, and in the presence ofoxygen, without destroying the activity for which the products areprepared. Further, with methods of the present invention, there is noneed to reduce the concentration of molecular oxygen.

The present invention contemplates that in vivo activity of a bloodproduct is not destroyed or significantly lowered if the blood productwhich is decontaminated by methods of the present invention tests aswould a normally functioning blood product in known assays for functionof the particular blood product. The activity of a clinical sample isnot destroyed or significantly lowered if the clinical sample which isdecontaminated by methods of the present invention tests as would anuntreated sample in common clinical chemistry tests. In contrast, ablood product or clinical sample is considered to have incurred“significant damage” when the blood product no longer functions for thepurpose it was prepared. For example, where red blood cells areconcerned, in vivo activity is not destroyed or significantly lowered ifATP levels, IgG binding, and extracellular potassium levels of the redblood cells are substantially the same in red blood cells treated by themethods of the present invention and stored 9 days as they are inuntreated samples stored for 9 days. Similarly, a clinical sample hasnot suffered significant damage if a treated sample tests substantiallythe same as an untreated sample in one or more common clinical chemistrytests. “Substantially the same” means that the values of the treatedsamples do not exhibit change which is more than 10% larger than changein values exhibited in a non treated control. In the case of red bloodcells, this comparison is made after a 9 day storage followingtreatment.

VI. PREPARATION OF VACCINES

The preparation of viral vaccines is also contemplated by methods of thepresent invention. The present invention contemplates producing vaccinesto a wide variety of viruses, including human viruses and animalviruses, such as canine, feline, bovine, porcine, equine and ovineviruses. The contemplated method is suitable for inactivating doublestranded DNA viruses, single stranded DNA viruses, double-stranded RNAviruses and single-stranded RNA viruses, including both enveloped andnon-enveloped viruses. A contemplated method for producing a vaccine forinoculation of a mammalian host susceptible to infection by a viruscomprises growing culture of virus, isolated from an infected host, in asuitable mammalian cell culture, exposing at least one of the seedviruses to a compound having a nucleic acid binding ligand and a mustardgroup for a time sufficient to inactivate the virus to a non-infectiousdegree, under conditions which substantially preserve the antigeniccharacteristics of the inactivated viral particles, and combining saidinactivated virus with a suitable adjuvant.

The inactivated virus may be formulated in a variety of ways for use asa vaccine. The concentration of the virus will generally be from about10⁶ to 10⁹ plaque forming units (pfu)/ml, as determined prior toinactivation, with a total dosage of at least 10⁵ plaque forming unitsper dose (pfu/dose), usually at least 10⁶ pfu/dose, preferably at least10⁷ pfu/dose. The total dosage will usually be at or near about 10⁹pfu/dose, more usually being about 10⁸ pfu/dose. The vaccine may includecells or may be cell-free. It may be an inert physiologically acceptablemedium, such as ionized water, phosphate-buffered saline, saline, or thelike, or may be administered in combination with a physiologicallyacceptable immunologic adjuvant, including but not limited to mineraloils, vegetable oils, mineral salts, and immunopotentiators, such asmuramyl dipeptide. The vaccine may be administered subcutaneously,intramuscularly, intraperitoneally, orally, or nasally. Usually, aspecific dosage at a specific site will range from about 0.1 ml to 4 ml,where the total dosage will range from about 0.5 ml to 8 ml. The numberof injections and their temporal spacing may be highly variable, butusually 1 to 3 injections at 1, 2 or 3 week intervals are effective.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); gm (grams); mg (milligrams); μg (micrograms); L (liters);ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters);μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); HPLC (HighPressure Liquid Chromatography); Q (quinacrine); QM (quinacrinemustard); DMSO (dimethylsulfoxide); Htc (hematocrit); RBC (red bloodcell); LB (Luria Broth); N-acetyl-cysteine (NAC); BUN (blood ureanitrogen); Creat. (creatinine); phos acid (phosphoric acid); alk(alkaline phosphatase); ALT (Alanine Aminotransferase); AST (AspartateTransaminase); LDH (lactate dehydrogenase); GGT (Glutamate Transferase);cfu (culture forming units); pfu (plaque forming units); DMEM(Delbecco's modified eagles medium); FBS (fetal bovine serum); PRBC(packed red blood cells); PCR (polymerase chain reaction); rpm(revolutions per minute); TC (tissue culture); NHSP (normal human serumpool); LSM (lymphocyte separation medium); NCS (newborn Calf Serum); PBS(phosphate buffered saline).

While it is available commercially from Baxter Healthcare Corp.,Deerfield, Ill., Adsol used in the following experiments was made bysterile filtering the following mixture: 22 g glucose, 9 g NaCl, 7.5 gmannitol, and 0.27 g adenine in 1 liter of distilled H20.

The polymerase chain reaction (PCR) is used in some of the examplesbelow. PCR is a method for increasing the concentration of a segment ofa target sequence in a mixture of genomic DNA without cloning orpurification. See K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and4,683,202, hereby incorporated by reference. This process for amplifyingthe target sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then areannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing, and polymerase extension can be repeated many times(i.e. denaturation, annealing and extension constitute one “cycle;”there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to by theinventors as the “polymerase chain reaction”.

EXAMPLE 1

This example measures the R17 inactivation activity of quinacrinemustard (QM) solutions made in either Adsol or DMSO. The bacteriophageR17 has a single stranded RNA genome of approximately 1.2×10⁶ daltons,and is difficult to inactivate compared to many other targets. Seegenerally L. Lin et al., Blood 74:517 (1989). The advantage of the R17system is that inactivation can be readily assayed in the laboratory.

The assay used to determine inactivation measures the ability of thephage to subsequently infect bacteria and inhibit their growth. Thephage was grown up in Hrf 3000 bacteria. (R17 and Hrf 3000 were obtainedfrom American Tissue Culture Collection (ATCC), Washington, D.C.).First, the R17 stock virus was diluted (10.9 logs/ml in LB broth) 1:20in Adsol (R17-Adsol). Then a 30% hematocrit (Htc) red blood cellconcentrate in R17-Adsol mixture was prepared by spinning down red bloodcells (RBC) from whole blood and resuspending 3.5 ml RBC pellet in 7.0ml R17-Adsol. In this, and the following experiments, Htc was measuredon a Model F800 Sysmex cell counter (Toa Medical Electronics, Kobe,Japan). Ten 1 ml aliquots of the samples were then transferred tosterile tubes.

Approximately 2 mg of QM, commercially available from Aldrich, Inc.,Milwaukee, Wis., was weighed out into each of two tubes. Samples werethen dissolved in DMSO or Adsol, respectively, to a final concentrationof 0.4 mg/ml. QM in Adsol is a suspension, not a solution, at thisconcentration.

Next, the QM suspension was added to the R17-Adsol samples to achievethe following final concentrations of QM in the sample tubes: 2.5, 5.0,10, or 20 μg/ml. The QM was completely solubilized at theseconcentrations. Positive control samples where also prepared, where 50μl of either Adsol or DMSO was added to R17-Adsol samples. The sampleswere allowed to stand at room temperature for at least 1 hour. Then thesamples were titered by an R17 phage assay. Sterile 13 ml dilution tubeswere prepares with LB broth. To make the dilutions, a 0.1 ml aliquot ofthe solution of phage was added to the first dilution tube of 0.4 ml ofmedia. Then 0.02 ml of this solution was added to the second tube of 0.5ml media (1:25). The second solution was then diluted serially (1:25)into the remaining tubes. To each diluted sample was added 0.05 ml ofHrf 3000 bacteria cultured overnight and 3 ml of molten LB top agar. Themixed materials were poured onto LB broth plates. After the top agarhardened, the plates were incubated at 37° C. overnight. Plaques werecounted the following morning and the titer of the phage remaining aftertreatment was calculated based on the dilution factors.

The results are shown in Table 2, below, and FIG. 1. It is dear from thedata that even at concentrations as low at 2.5 μg/ml QM is effective ininactivating R17. At concentrations above 10 μg/ml, completeinactivation is achieved, to the limit of detection of assay.

TABLE 2 Sample # (QM) (μg/ml) Solvent Log Titer 1 0 Adsol 9.8 2 0 DMSO9.8 3 2.5 Adsol 3.25 4 5 Adsol 3.55 5 10 Adsol 1.0 6 20 Adsol 1.3 7 2.5DMSO 5.2 8 5 DMSO 2.3 9 10 DMSO 2.4 10 20 DMSO 1.0

EXAMPLE 2

The purpose of this example is to show that the presence of RBC does notsignificantly effect R17 inactivation by compounds and methods of thepresent invention. Two different compounds were tested, QM and Compound1, the synthesis of which is described in Example 17, below. For QM, theprocedure was as follows: first, approximately 60% Htc RBC concentratewas prepared by dilution in Adsol. The sample was again diluted withAdsol into sterile tubes to give RBC concentrate with a final Htc of 2%,6%, 20% or 60% (0.5 ml final volume in each tube).

Next, an R17 stock (11.3 logs/ml in LB) was diluted 1:10 in Adsol(R17-Adsol). This stock was added (0.5 ml) to each tube to give a finalHtc of approximately 1%, 3%, 10% or 30% in 1 ml. A positive controlsample was prepared without RBC by combining 0.5 ml of R17-Adsol with0.5 ml Adsol. QM (3.4 mg) was dissolved in H₂O to reach a finalconcentration of 0.1 mg/ml. Then a 10 μl aliquot of the QM solution wasadded to each R17 sample and the samples were incubated approximately 2hours. A negative control was not treated with QM. The samples were thentitered in an R17 phage assay, as described in Example 1, above.

The results are shown in Table 3 and FIG. 2. It is clear from the datathat QM inactivates R17 in all of the Htc tested.

TABLE 3 Sample # Htc (%) QM (μg/ml) Log Titer 1 0 0 8.8 9 0 1.0 2.0 10 11.0 4.0 11 3 1.0 2.1 12 10 1.0 2.6 13 30 1.0 2.4

Another experiment was performed to test the inactivation ability of anovel compound, Compound 1. A 1:1000 dilution of R17 (stock titer was11.9 logs) was prepared in 25 ml packed red blood cells. To each of 5tubes was added 5 ml of this R17-packed red blood cell solution.Compound 1 was then dissolved in saline to a final concentration of 3mg/ml. The compound in solution was added to the 4 tubes as follows: thefirst tube, the control tube, received saline only; the second tubereceived 10 μg/ml of Compound 1 in saline; the third tube received 30μg/ml of Compound 1 in saline; the forth tube received 100 μg/mlCompound 1 in saline and the final tube received 300 μg/ml of Compound 1in saline. The tubes were mixed and then incubated at 4° C. overnight.The results showed R17 inactivation activity. Concentrations above 30μg/ml inactivated approximately 4 logs of R17 with a starting titer of10 logs of R17.

EXAMPLE 3

This example sets forth the kinetics of R17 inactivation by QM. Tomeasure the kinetics of inactivation, reactive QM must be quenched sothat intermediate time points provide a reliable measure of the R17inactivation at a particular time. Two methods were used here incombination to quench the reaction. First, NAC was added to samples toreact with excess QM. Second, samples were rapidly diluted into LBmedium to reduce the effective QM concentration in the sample. Thecontrol experiments described below demonstrate that this dual approachdoes effectively quench residual QM, allowing for a valid measure of thereaction kinetics to be taken.

Samples were prepared in the following manner. A dilution of R17 (1:20)into Adsol was prepared: 0.15 ml phage (11.3 logs/ml)+2.85 ml Adsol. Analiquot of sterile-filtered 0.1 M NAC was thawed for use to quench theQM reaction with R17.

Tubes were then prepared for standard dilution of phage, containingappropriate volumes of LB.

TABLE 4 Sample # Treatment 1 QM first, NAC quench at 0 min., dilute 2 QMfirst, NAC quench at 2 min., dilute 3 QM first, NAC quench at 4 min,dilute 4 QM first, NAC quench at 8 min, dilute 5 QM first, NAC quench at16 min, dilute 6 QM first, NAC quench at 32 min, dilute 7 QM first, NACquench at 64 min, dilute 8 QM first, NAC quench at 128 min, dilute 9first add NAC, then QM, dilute 10 first add NAC, then QM, dilute at end11 add NAC/no QM, dilute 12 add NAC/no QM, dilute at end 13 no NAC/noQM, dilute at end

A set of tubes were prepared, herein called quenching tubes, containingquenching factors (NAC and/or dilution with LB), to receive the samplesat appropriate time points. Cysteine (44 μl aliquots) was added toquenching tubes numbered 1-2.

QM (1.5 mg) was dissolved in Adsol (25.0 ml) to a final concentration of0.1 mg/ml. Then the QM solution was diluted 100× into Adsol: 50 μl QMsolution+4.95 ml Adsol; 1 μg/ml final concentration.

Table 4 sets forth how each control and experimental sample was treated.The control where treated first, by placing aliquots (100 μl) of phageinto quenching tubes 9-13, then immediately adding 100 μl of 1 μg/ml QMto quenching tubes 9 and 10 and 200 μl Adsol to quenching tubes 11 and12. Adsol (250 μl) was added to quenching tube 13. Then samples 9 and 11were diluted into LB broth for phage assay.

The experimental samples were treated next. Phage (1.0 ml) was removedinto a sterile 15 ml tube. QM (1.0 ml, 1.0 μg/ml) was added. Thismixture was removed (by 200 μl aliquots) into quenching tubes 1-8 at thefollowing times: 0, 2, 4, 8, 16, 32, 64, and 128 minutes. The sampleswere mixed and immediately diluted into LB broth for phage assay.Finally, samples 10, 12, and 13 were diluted into LB broth for phageassay.

Results are shown in Table 5 and FIG. 3. While NAC alone does not killR17 (compare samples #11 and #12 with sample #13), when added before QM,NAC provided a substantial, but not complete protection against QMinactivation (compare samples #7 and #10). The combination of NAC anddilution resulted in almost complete quenching of QM activity (comparesamples #1 and #13). QM inactivation of R17 was complete within 2 hours.

TABLE 5 Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 Log Titer 8.11 6.81 5.794.10 3.74 2.69 2.39 ≦2.4 8.66 6.32 8.73 8.35 8.80

EXAMPLE 4

This experiment measures the loss of QM activity upon pre-incubation ofdrug in Adsol. It is believed that mustards react by thermally allowedpathways. They can be hydrolyzed in aqueous solution. This experimentwas designed to measure loss of QM anti-viral activity in a particularaqueous solution, Adsol. Previous results have shown that QM anti-viralactivity did not decrease rapidly upon pre-incubation of the drug inAdsol. (Results not shown). A concern in those experiments was thepossibility of light-dependent inactivation, because samples werediluted into LB without making extraordinary efforts to shield ambientlight, and because acridines are known to inactivate by photodynamiceffects. This experiment was repeated under conditions where ambientlight levels were carefully controlled throughout the experiment, inorder to exclude the possibility that R17 inactivation was due tolight-mediated effects. Also, additional controls were added to examinethe effects of light in samples that were deliberately exposed to roomlights and to examine the inactivation by the parent compound,quinacrine, the structure of which follows:

The following procedure was performed in a biosafety cabinet withoutlights. R17 phage (10.9 logs/ml) was diluted 10 fold into Adsol: 1.0 mlR17 stock+9.0 ml Adsol. One (1) ml of the diluted phage was transferredinto ten sterile 1.5 ml tubes. A 0.1 mg/ml QM solution was prepared bydissolving 3.4 mg QM (weighed in hood) with 34 ml H₂O. The resultingsolution was wrapped in foil to shield from light. Then, 10 μl QM wasadded to tubes after 0, 10, 40, 60, 120 or 240 minutes ofpre-incubation. Samples were again wrapped in foil to prevent exposureto light. For a light control, 10 μl QM was added to 1.0 ml phage attime 0, and the sample was not wrapped in foil. A 1 mg/ml solution ofquinacrine in DMSO was prepared as another control. One (1) μl of thiswas added to each of two samples. Then one sample was incubated in foil(sample Q) and one without foil (sample Q+light). All samples wereincubated for 2 hours 15 minutes beyond final addition of QM. Totalincubation for the time zero sample was 6 hours 15 minutes.

The following work was performed with very low ambient light (source wasone closed doorway): bacteria was diluted and plated in the dark. Forthe light positive controls, the samples were exposed to ambient lightduring dilutions, then moved to dim lighting conditions during plating.

The results are shown in FIG. 4 and Table 6. From these results it isclear that there was no light-dependent kill by QM or quinacrine underthe conditions of this experiment. Further, QM activity was notdiminished after a 4 hour pre-incubation in Adsol.

TABLE 6 QM + Q + Sample Control light Q light 0 min 10 min 40 min 1 hour2 hour 4 hour Log 9.7 3.9 9.7 9.7 3.8 3.9 3.9 4.0 4.8 4.0 Titer

EXAMPLE 5

QM activity was not diminished after a 4 hour pre-incubation in Adsol,as shown by Example 4, above. A goal of this example is to determinewhether QM is inactivated more rapidly by pre-incubation in the presenceof red cells. This example also examines the kinetics of removal of QMfrom red blood cell solutions by an adsorbent material, to establish theeffectiveness of a scrub technique in removing compounds containing amustard group.

First, phage dilutions were prepared. R17 (11.3 log/ml stock) wasdiluted 1:10 into Adsol,: 0.7 ml phage+6.3 ml Adsol. Diluted phage (0.5ml) was placed into 15 sterile 1.5 ml tubes labeled 1-15. The treatmentfor each tube is shown in Table 7.

TABLE 7 Time Sample # Treatment (min) 1 none — 2 QM-adsol 0 3 QM-adsol240 4 QM-RBC 0 5 QM-RBC 15 6 QM-RBC 30 7 QM-RBC 60 8 QM-RBC 120 9 QM-RBC240 10 QM-XAD 0 11 QM-XAD 15 12 QM-XAD 30 13 QM-XAD 60 14 QM-XAD 120 15QM-XAD 240

Next, QM solutions were prepared. Approximately 20 ml of packed redblood cells (PRBC) were spun down in a 50 ml conical tube at 1600 rpmfor 9 minutes. The volume of the pellet after spinning was 17 ml.Approximately 3 mg QM was weighed out on a weighing paper in a biosafetycabinet (actual weight was 4.5 mg). The sample was then transferred to a50 ml conical tube. The sample was dissolved in Adsol to a concentrationof 0.1 mg/ml (actual volume of Adsol was 45 ml). Next, the red bloodcell pellet was diluted 1:1 with 17 ml of the QM solution. The tubecontents were mixed gently by inversion several times. This issubsequently called the QM-RBC solution.

Amberlite XAD 16™ (a commercially available adsorbent from Sigma, St.Louis, Mo.) was weighed out (0.452 g) and transferred to a 15 ml conicaltube. An aliquot of the QM-RBC solution (9 ml) was transferred to the 15ml tube containing 0.5 g XAD-16 and mixed gently by inversion. This issubsequently referred to as the QM-XAD solution. The QM solution wasdiluted with an equal volume of Adsol (1 ml of each). This issubsequently referred to as the QM-Adsol solution.

At each time point, 0.1 ml was removed from QM-RBC, QM-XAD and QM-Adsolinto a 1.5 ml Eppendorf tube. The tubes contents were spun down 10 secat full speed in a microfuge to pellet cells and resin. Then 5 μlaqueous phase containing QM was transferred to the appropriate tubecontaining phage. The phage containing QM was then incubated in the darkfor the times specified in Table 7, above. The 240 minute sample wasincubated at least 2 hours after addition of QM. Finally, dilutions weremade and the phage were plated.

The results appear in Table 8, below, and FIG. 5. QM anti-viral activitywas removed upon a 4 hour pre-incubation with red blood cells. Theadsorbent scrub material, Amberlite XAD-16™, also removed QM from bloodwithin 1 hour. These results suggest that either incubation in thepresence of red blood cells or treatment with an adsorbent resin, or thetwo treatments combined, will be sufficient to rapidly remove residualQM after inactivation.

TABLE 8 Sample Log Titer 1 9.7 2 2.9 3 3.7 4 2.8 5 3.4 6 5.0 7 7.1 8 8.09 9.7 10 3.2 11 7.9 12 9.1 13 9.6 14 9.8 15 9.8

EXAMPLE 6

The purpose of this example is to measure inactivation of duck hepatitisB virus (DHBV) by a method of the present invention. DHBV was chosen asa model for human hepatitis B virus because of the similarities indesign between the two viruses. See Ganem, D. and Varmus, H. “TheMolecular Biology of the Hepatitis B Viruses,” Ann. Rev. 56:651 (1987).

Infected duck hepatocytes were prepared as follows. Duck hepatocyteswere isolated from the livers of approximately 1 week old ducklings.Ducklings were prescreened and found negative for DHBV. Each of theducklings was anesthetized, then infused with 0.5 ml sodium heparin viathe portal vein. Next, each duckling was perfused with 75 ml of asolution containing 200 ml 1× MEM/Earle's BSS+2 ml Hepes buffer+2 ml of1% EGTA (in 1× MEM). Then, the ducklings were perfused for 20 minuteswith a filter sterilized solution containing 30 mg of Collagenase A(commercially available from Boehringer-Mannheim Biochem., Indianapolis,Ind.)+200 ml Ham's F-12/DMEM medium.

At this point, the liver was removed, cut up into a fine mush and placedin a 125 ml bottle containing 50 ml Ham's F-12/DMEM. Approximately 10 mlof a solution containing 5 mg DNase I and 25 ml Ham's F-12/DMEM wasadded to the liver suspension. The suspension was spun at 200 rpm for 10minutes.

The suspension was then strained through gauze pads, the 125 ml bottlewas rinsed with the remaining 15 ml of the DNase I solution and therinsing was also strained into the liver suspension. The cell suspensionwas equally divided into 2×50 ml centrifuge tubes and pelleted at 50×gfor 2 minutes. The pellets were resuspended in 10 ml of a solutioncontaining Medium 199/Earle's BSS, 5% calf serum and pelleted. Thisprocess was repeated two more times. The third pelleting was resuspendedin 10 ml plating medium. Another 10 ml plating medium was added to eachtube.

The liver cell suspension was filtered through a 70 micron cell strainerinto a 50 ml centrifuge tube. Aliquots of the cell suspension(approximately 0.5 ml) were transferred to petri dishes containing 2 mlplating medium, to obtain a level of confluency corresponding toapproximately 6 to 8×10⁶ viable cells per petri dish. After a two hourincubation at 37° C., the medium was changed to L-15 medium(commercially available from Gibco, Grand Island, N.Y.) (containing 0.9g/L galactose, 0.55 g/L Na pyruvate)/DMSO. The medium was again changedat 24 hours and every 48 hours thereafter. Cells were grown in culturefor 5-7 days.

Next, viral inactivation was performed. DHBV stock virus was thawed at37° C. for 15 min in an oven. The virus was then spun down at 14000 rpmin a microfuge for 5 min at room temperature and the supernatant wastransferred to a fresh tube, avoiding material at the bottom of thetube. The spin and transfer were repeated and the samples were placed onice.

Approximately 7 ml of whole blood was drawn into a tube containing acidcitrate dextrose anticoagulant. The cells were spun down at 1600 rpm for9 minutes to pack the red cells. The plasma was withdrawn and replacedwith an equal volume of Adsol (2.9 ml).

The virus was diluted 0.25 ml into 2.25 ml red blood cells and themixture was vortexed to create a 10⁻¹ dilution. The diluted virus wasthen aliquoted in sterile tubes as follows: 50 μl as an untreatedsample; 1.8 μl to be treated with 40 μg/ml QM; and 0.5 ml to be treatedwith 10 μg/ml QM. Next, a 1 mg/ml QM solution was prepared by dissolving3.2 mg QM in 3.2 ml sterile ddH₂O. Aliquots of the QM solution wereadded to the tubes containing virus, as follows: 72 μl QM was added tothe 1.8 ml sample to achieve a final concentration of QM of 40 μg/ml and5 μl QM was added to the 0.5 ml sample to achieve a final concentrationof QM of 10 μg/ml. The samples were incubated for 4 hours at roomtemperature.

After incubation the red blood cells were spun down in a microfuge.Plasma/Adsol supernatant was removed. Dilutions of each sample wereprepared by serial dilution of 100 μl virus into 0.9 ml PBS/10% NCS (PBSwas 10 mM, pH 7.4). The untreated control (sample #1) was diluted to10⁻⁷. The treated samples (#2 and #3) was diluted to ₁₀ ⁻⁴.

Plates containing liver suspension were then inoculated according to thescheme set forth in Table 9. Plates were inoculated with approximately100 μl virus in duplicate (see below) and cultured for 1, 10, or 15days. Samples were then analyzed by PCR and by slot blot hybridizationto confirm the presence of viral DNA.

Slot blot hybridization was performed for all of the samples afterharvesting DNA from tissue culture samples. PCR analysis was performedon selected samples. Samples were denatured with 3M NaOH, as wereplasmid pD1.5G DNA samples for labeling. Samples were then neutralizedwith NH₄OAc. 400 μl of 1M NH₄OAc was added to each well of a Mini FoldII Slot Blot Apparatus, commercially available from VWR Scientific,Greenbelt, Mo., fitted with a filter, as were aliquots of each sample.Vacuum was applied to the apparatus until all samples had been pulledthrough the filter. The filter was then baked to dry. Next, the filterwas pre-hybridized in a mixture of 250 ml of 20×SSC (175.3 g NaCl, 88.2g Na citrate in 800 ml H₂O), 50 ml of 50× Denhardt's solution (5 gFicoll, available from Sigma, St. Louis, Mo., 5 g polyvinylpyrrolidone,and 5 g bovine serum albumin with 500 ml H₂O), 20 ml of h mg/mldenatured salmon sperm DNA, 180 ml H₂O, 500 ml formamide and 10 ml of10% solution of sodium dodecyl sulfate in H₂O. Probe was prepared asfollows: 3 μl of pD1.5G (67 ng/μl) and 5 ml of 15 ng/μl random hexameroligonucleotides were heated and cooled again, then 4 μl of 5× labelingbuffer, 2 μl of dGAT mixture (5 mM each of dGTP, dATP, dTTP, in TE), 1μl of Klenow, and 5 μl of [a³²P]dCTP was added and incubated. Reactionwas stopped by adding 25 mM EDTA. Then 5×10⁵ counts per minute of probeper ml of hybridization solution was added to the filter and allowed tohybridize overnight. The filter was removed, and low stringency washsolution (50 ml of 20×SSC, 940 ml of H₂O, and 10 ml of 10% SDS) wasadded to cover the filter for a wash during shaking, which was repeat 2times, the last time adding high stringency was solution (5 ml of20×SSC, 990 ml of H₂O, and 10 ml of 10% SDS) instead. The filter wasthen exposed to film to obtain an appropriate exposure, and the film wasthen scored for positive hybridization. A negative control samplecontaining calf thymus DNA was also run.

TABLE 9 Sample Treatment Dilution Incubation 1 no virus NA 10 days 2untreated 10⁻⁷ 10, 15 3 untreated 10⁻⁶ 10, 15 4 untreated 10⁻⁵ 10, 15 5untreated 10⁻⁴ 10, 15 6 untreated 10⁻³ 10, 15 7 40 μg/ml QM 10⁻⁵ 10, 158 40 μg/ml QM 10⁻⁴ 10, 15 9 40 μg/ml QM 10⁻³ 10, 15 10 40 μg/ml QM 10⁻²10, 15 11 40 μg/ml QM 10⁻¹  1, 10, 15 12 10 μg/ml QM 10⁻⁵ 10, 15 13 10μg/ml QM 10⁻⁴ 10, 15 14 10 μg/ml QM 10⁻³ 10, 15 15 10 μg/ml QM 10⁻²  1,10, 15

Table 10 summarizes PCR and slot blot hybridization data. (NP signifiesthat PCR was “not performed” for that sample. A plus sign signifies thatDHBV nucleic acid was amplified in PCR. A minus sign signifies that itwas not amplified).

TABLE 10 Sample # Incubation (days) Plate #'s Blot Results PCR Results 110  1*, 2 −, − − 2 10  3, 4 −, − NP 2 15  5*, 6 −, − − 3 10  7, 8 −, −NP 3 15  9*, 10 −, − + 4 10 11*, 12 −, − − 4 15 13*, 14 ±, + + 5 10 15*,16* +, − +, + 5 15 17*, 18 +, + + 6 10 19, 20* +, + + 6 15 21*, 22+, + + 7 10 23, 24 −, − NP 7 15 25, 26 −, − NP 8 10 27, 28 −, − NP 8 1529, 30 −, − NP 9 10 31*, 32 −, − − 9 15 33*, 34 −, − − 10 10 35*, 36 −,− − 10 15 37*, 38 −, − − 11 1 39, 40* −, − − 11 10 41*, 42 −, − − 11 1543*, 44 −, − − 12 10 45, 46 −, − NP 12 15 47, 48 −, − NP 13 10 49, 50*−, − − 13 15 51, 52 −, − NP 14 10 53*, 54* −, − −, − 14 15 55, 56* −, −− 15 1 57*, 58 −, − − 15 10 59, 60* −, − + 15 15 61*, 62 −, − + *Theseplates were tested in PCR. Results appear in PCR column.

Referring to Table 10, viral titer was 6 logs per ml based on PCRpositive signal for plate #9. A dose of 10 μg/ml QM inactivated 4 logsper ml based on PCR positive signal for plates #60 and #61. A dose of 40μg/ml QM inactivated >6 logs of DHBV per ml based on the absence of aPCR signal and slot blot signals in all samples tested.

EXAMPLE 7

The purpose of this example is to measure inactivation of cell-free HIVby QM. As in the R17 assay, small aliquots of QM were added to stockHIV-1. The stock QM solution was prepared by dissolving 3.4 mg of thecompound in tissue culture media (DMEM/15% FBS) to reach a finalconcentration of 0.6 mg/ml of QM. The QM was a colloidal suspensionrather than a solution at this concentration, which was used in theexperiment. Stock HIV (10^(4.2) plaque forming units/ml) was in DMEM/15%FBS. QM solution was added to aliquots of stock HIV-1 to obtain a finaltotal sample volume of 0.5 ml, having the following final concentrationsof QM: 3 μg/ml, 10 μg/ml, or 30 μg/ml. The 0.5 ml test aliquots wereplaced in 24 well polystyrene tissue culture plates. Two controls wereprepared, one containing HIV-1 stock only, and one containing QM withoutHIV-1 stock. All samples were incubated for one hour at roomtemperature, then stored at −70° C. until assayed for infectivity by amicrotiter plaque assay. Aliquots for measurement of residual HIVinfectivity in the samples treated with a compound of the presentinvention were withdrawn and cultured.

Residual HIV infectivity was assayed using an MT-2 infectivity assay.(Previously described in Hanson, C. V., Crowford-Miksza, L. andSheppard, H. W., J. Clin. Micro 28:2030 (1990)). The assay medium was85% DMEM (with a high glucose concentration) containing 200 μg ofstreptomycin, 200 U of penicillin, 50 μg of gentamicin, and 1 μg ofamphotericin B per ml, 15% FBS and 2 μg of Polybrene (Sigma ChemicalCo., St. Louis, Mo.) per ml. Test and control samples from theinactivation procedure were diluted in 50% assay medium and 50% normalhuman pooled plasma. The samples were serially diluted in 96-well plates(Corning Glass Works, Corning, N.Y.). The plates were incubated at 37°C. in a 5% CO₂ atmosphere for 1 to 18 hours. MT-2 cells (0.025 ml)[clone alpha-4, available (catalog number 237) from the NationalInstitutes of Health AIDS Research and Reference Reagent Program,Rockville, Md.] were added to each well to give a concentration of80,000 cells per well. After an additional 1 hour of incubation at 37°C. in 5% CO₂, 0.075 ml of assay medium containing 1.6% SeaPlaque agarose(FMC Bioproducts, Rockland, Me.), prewarmed to 38.5° C. was added toeach well. The plates were kept at 37° C. for a few minutes untilseveral plates had accumulated and then centrifuged in plate carriers at600×g for 20 minutes. In the centrifuge, cell monolayers formed prior togelling of the agarose layer. The plates were incubated for 6 days at37° C. in 5% CO₂ and stained by the addition of 0.05 ml of 50 μg/mlpropidium iodide (Sigma Chemical Co.) in phosphate-buffered saline (pH7.4) to each well. After 24 to 48 hours, the pink/orangefluorescence-stained microplaques were visualized by placing the plateson an 8,000 μW/cm² 304 nm UV light box (Fotodyne, Inc., New Berlin,Wis.). The plaques were counted at a magnification of 20× to 25× througha stereomicroscope.

TABLE 11 Sample Log Titer no QM 4.2  3 μg/ml QM 3.4 10 μg/ml QM 2.0 30μg/ml QM <1.7

The results appear in Table 11, above. At a concentration of 30 μg/ml,QM was able to inactivate cell-free HIV completely to the level ofdetection of the plaque assay used.

EXAMPLE 8

The last example demonstrated that QM was able to inactivate cell freeHIV. HIV can also be found within certain types of cells. This exampleexamines the ability of QM, at varying concentrations, to inactivate thecell-associated form of HIV.

H9 cells chronically infected with HIV_(IIIB) were used. (H9/HTLV-III-BNIH 1983 Cat.#400). Cultures of these cells were maintained in highglucose Dulbecco Modified Eagle Medium supplemented with 2 mML-glutamine, 200 units/ml penicillin, 200 μg/ml streptomycin, and 9%fetal bovine serum (Intergen Company, Purchase, N.Y.) For maintenance,the culture was split once a week, to a density of 3×10⁵ to 4×10⁵cells/ml. About four days after splitting, 8.8% sodium bicarbonate wasadded as needed. For the inactivation procedure, the cells were usedthree days after they were split. They were spun from their culturemedium at 400 g for 10 minutes, the supernatant was discarded, and thecells were resuspended in approximately 8 ml of 85% DMEM+15% FBS, to aconcentration of 2×10⁶ cells/ml. Aliquots (1 ml) of the infected cellsuspension were placed in 15 ml tubes for QM free controls and for theQM experimental sample. A stock solution of QM (1 mg/ml in sterileddH₂O) was diluted into the 15 ml tubes in the appropriate aliquots toyield a final concentration of either 0, 3, 10, 30, 100, or 150 μg/ml.The samples were incubated for two hours, with periodic thorough mixing,then stored at −80° C. until analyzed by microtiter plaque assay.

The stored samples were thawed at 37° C., then titrated in an HIVmicrotiter plaque assay, as described in Hanson, C. V., Crawford-Miksza,L. and Sheppard, H. W., J. Clin. Micro 28:2030 (1990), and as describedin Example 7, above, with the following modifications. The samples wereserially diluted directly in 96-well plates (Corning Glass Works,Corning, N.Y.). The plates were incubated at 37° C. in a 5% CO₂atmosphere for 1 to 18 hours. MT-2 cells (0.025 mL) [clone alpha-4,available (catalog number 237) from the National Institutes of HealthAIDS Research and Reference Reagent Program, Rockville, Md.] were addedto each well to give a concentration of 80,000 cells per well. After anadditional 1 hour of incubation at 37° C. in 5% CO₂, 0.075 mL of assaymedium containing 1.6% SeaPlaque agarose (FMC Bioproducts, Rockland,Me.), prewarmed to 38.5° C. was added to each well. The plates were keptat 37° C. for a few minutes until several plates had accumulated andthen centrifuged in plate carriers at 600×g for 20 minutes. In thecentrifuge, cell monolayers formed prior to gelling of the agaroselayer. The plates were incubated for 6 days at 37° C. in 5% CO₂ andstained by the addition of 0.05 mL of 50 μg/mL propidium iodide (SigmaChemical Co.) in phosphate-buffered saline (pH 7.4) to each well. After24 to 48 hours, the pink/orange fluorescence-stained microplaques werevisualized by placing the plates on an 8,000 μW/cm² 304 nm UV light box(Fotodyne, Inc., New Berlin, Wis.). The plaques were counted at amagnification of between 20× and 25× through a stereomicroscope.

The results appear in Table 12.

TABLE 12 Sample Log Titer Log Reduction no QM 5.5 —  3 μg/ml 2.7 −2.8 10 μg/ml <0.7 > −4.8  30 μg/ml <0.3 > −5.2 100 μg/ml 1.75 −3.75 150μg/ml <0.3 > −5.2

It is clear from this example that QM inactivates cell-associated HIV,even at very low concentrations such as 10 μg/ml and below.

EXAMPLE 9

This example sets forth the ability of two compound having a nucleicacid binding ligand and a mustard group, QM, andN-(2-chloroethyl)-N-ethyl-N′-(6-chloro-2-methoxy-9-acridinyl)-1,3-propanediaminedihydrochloride (“ICR-170”) (commercially available from PolysciencesInc, Warrington, Pa.) to inactivate both cell-free and cell-associatedHIV in the presence of red blood cells. The structure of ICR-170 isshown below.

For the cell free HIV inactivation, 15 ml of PRBC was mixed with 5 mlAdsol for a final volume of 20 ml. Then ten 2 ml aliquots were added to15 ml conical tubes. Varying doses of the two compounds were next addedto the tubes. The stock compound solutions were both 1 mg/ml in saline,stored at 4° C. ICR-170 was in solution at this concentration. Thefollowing volumes of the two test compounds were added to the PRBCtubes: 20, 40, 80 or 160 μl; to produce final concentrations of the testcompound of 10, 20, 40, or 80 μg/ml.

After addition of the compounds, the samples were incubated for 100minutes at room temperature in the dark, with mixing every 30 minutes.Subsequently, the red blood cells were pelleted by spinning for 5minutes at 2500 rpm. The supernatant was removed and NHSP was added sothat the sample contained 15% NHSP. Samples were stored at −80° C.

Inactivation of cell-associated HIV was performed in a similar manner,with the following exceptions. H9 cells chronically infected withHIV_(IIIB) were used. (H9/HTLV-III-B NIH 1983 Cat.#400). Cultures ofthese cells were maintained in high glucose DMEM supplemented with 2 mML-glutamine, 200 units/mL penicillin, 200 μg/ml streptomycin, and 9%fetal bovine serum (Intergen Company, Purchase, N.Y.) For maintenance,the culture was split once a week, to a density of 3×10⁵ to 4×10⁵cells/ml and about four days after splitting, 3.3% sodium bicarbonatewas added as needed. For the inactivation procedure, the cells were usedthree days after they were split. The cells in a sample of this stockwere counted on a Neubauer type Hemacytometer (commercially availablefrom VWR Scientific, Greenbelt, Mo.), and found to have 1.07×10⁶cells/ml. An aliquot (18.7 ml, 20×10⁶ cells) was pelleted andresuspended in 5 ml Adsol. This 5 ml of cell suspension was then addedto 15 ml of PRBC. The sample was divided and compound was added asdescribed above for the cell-free samples. The samples were incubatedfor 100 minutes, followed by the addition of 3 ml of a 1:1 mixture ofNHSP and RPMI-1640 (commercially available from Irvine Scientific, SantaAna, Calif.). Next, each sample was placed in a 15 ml tube containing 6ml lymphocyte separation medium (LSM) (commercially available fromOrganon Teknika Corp., Durham, N.C.) and the tubes were spun at 1500 rpmfor 30 minutes. The H9 cells, which separated into a distinct layer,were removed to another tube, mixed with 10 ml DMEM and spun at 2000 rpmfor 5 minutes. The pellet was resuspended into 1 ml of 85% DMEM+15% FBS,and then transferred to a 2 ml sarstedt tube. The samples were alsostored at −80° C.

The samples were titered using a microtiter plaque assay, as describedin Example 8 for cell-free HIV and Example 9 for cell-associated HIV.The results appear in Table 13A (cell free) and 13B (cell associated),below.

TABLE 13A Cell-free HIV Inactivation Log Titer Compound Concentration(μg/ml) pfu/ml Log Reduction QM 0 5.7 — 10 3.9 1.8 20 3.0 2.7 40 0 >4.380 0 >4.3 ICR-170 0 5.7 — 10 5.1 0.6 20 4.4 1.3 40 3.4 2.3 80 1.7 4.0

TABLE 13B Cell-associated HIV Inactivation Compound Concentration(μg/ml) Log Titer Log Reduction QM 0 5.5 — 10 4.4 1.1 20 3.6 1.9 40 2.53.0 80 2.4 3.1 ICR-170 0 5.7 — 10 5.2 0.5 20 4.5 1.2 40 3.7 2.0 80 3.72.0

EXAMPLE 10

The above examples have established that QM has exceptional pathogeninactivation activity. In choosing an agent to decontaminate bloodproducts for clinical testing or transfusion, it is also important toconsider the effects of the method and compound used on blood productfunction. This example explores the short term effects of two compounds,one having a nucleic acid binding ligand and a mustard group, QM andchlorambucil on red blood cell function, as measured by potassiumleakage and IgG binding to red blood cell surfaces. The structure ofchlorambucil appears below.

This example additionally compares the R17 inactivation activity, in redblood cells, of a compound having both a nucleic acid binding ligand anda mustard group (QM), with a compound having only a mustard group, andno nucleic acid binding ligand (chlorambucil).

Whole blood (20 ml) was transferred to a 50 ml conical tube and spundown at 1600 rpm for 9 minutes at room temperature. Plasma was removed(9 ml). Next, 10.9 logs/ml stock of R17 phage was diluted 1:20 withAdsol (24.4 ml Adsol+1.28 ml R17). The pelleted red blood cells werethen resuspended to 30% Htc with 25.6 ml of the Adsol/R17 mixture.Aliquots (3 ml each) were transferred into 9 tubes on ice.

Each mustard was added to Adsol. Chlorambucil, commercially availablefrom Aldrich Inc., Milwaukee, Wis., (5.8 mg) was added to 1.93 ml Adsolplus 5.85 μl 3M NaOH (undissolved material remained, and suspension wasused in the experiment by swirling before addition. QM (2.9 mg) wasadded to 0.967 ml Adsol (again, material remained in suspension). Themustards were immediately added to the blood, at volumes set forth inTable 14, below, and mixed by inversion.

TABLE 14 Sample Contents Volume Mustard 1 control none 2  10 μg/mlChlorambucil  10 μl 3  30 μg/ml Chlorambucil  30 μl 4 100 μg/mlChlorambucil 103 μl 5 300 μg/ml Chlorambucil 333 μl 6  10 μg/mlQuinacrine  10 μl 7  30 μg/ml Quinacrine  30 μl 8 100 μg/ml Quinacrine103 μl 9 300 μg/ml Quinacrine 333 μl

Extracellular potassium levels were measured approximately one hourafter treatment using a Ciba Corning 614 K⁺/Na⁺ Analyzer (commerciallyavailable from Ciba Corning Diagnostics Corp., Medfield, Mass.). Theremaining samples were incubated overnight at 4° C. After incubation,0.2 ml of each sample was removed for R17 assay and spun in a microfugefor 1 min. Supernatant was then removed for phage assay.

Potassium levels on remaining samples were measured and the samples werestored at 4° C. Potassium measurements were repeated daily for one weekor until significant differences were observed. Extracellular potassiumdata appears in Table 15, and FIG. 6. IgG binding in the samples wasmeasured using Baxter Unival Anti-Human Globulin Anti-IgG for DirectAntiglobulin Test and Baxter Coombs control Cells for Quality Control ofAnti-Human Globulin Test (both available from Baxter HealthcareCorporation, Deerfield, Ill.). The results of IgG Binding as measured byFACScan™ (Becton Dickinson, Mountain View, Calif.) appear in Table 16.

R17 was completely inactivated at all concentrations of QM (≧8.4logs/ml). However, little or no inactivation (≦0.4 logs) was observedfor Chlorambucil, up to a concentration of 300 μg/ml.

TABLE 15 Extracellular Potassium (mM) Day Day Day Day Day Day Day Sample0 1 2 3 4 6 7 1 0.70 1.59 2.51 3.16 3.71 4.63 5.01 2 0.76 1.59 2.45 3.183.72 4.57 4.94 3 0.69 1.56 2.40 3.16 3.72 4.69 5.03 4 0.72 1.74 2.433.18 3.76 4.73 5.12 5 0.72 1.71 2.58 3.31 3.92 4.89 5.36 6 0.73 1.652.76 3.64 4.26 5.30 5.69 7 0.76 1.94 3.08 4.00 4.63 5.76 6.15 8 0.782.48 4.05 5.23 6.06 7.50 8.02 9 0.82 3.61 5.59 7.37 8.32 >10 11.20

TABLE 16 Sample 1 2 3 4 5 6 7 8 9 Median 3.43 3.31 3.37 3.31 3.62 5.007.77 15.8 48.7 Fluor- escence

Chlorambucil did not alter potassium leakage or IgG binding of redcells. QM showed significant anti-viral activity and only induced slightchanges in red blood cell function under the conditions of thisexperiment. Significant red blood cell damage was only detected atlevels far higher than that required to inactivate R17.

EXAMPLE 11

Example 10 showed that QM was able to inactivate R17 in red blood cellsunder conditions where potassium leakage and surface IgG binding werenegligible. This example is designed to further these observations bylooking more extensively at red blood cell function after treatment withvarying levels of QM. Specifically, this example looks at the effects ofQM treatment on red blood cell function after storage under conditionsthat closely mimic those in a blood bank.

A packed red blood cell unit, approximately 1 day old, was obtained fromSacramento Blood Center. The cells were resuspended and approximately200 ml was transferred to a sterile container. R17 (0.2 ml) in LB wasadded and the sample was mixed. Next the unit was divided into 6-30 mlaliquots in sterile conical centrifuge tubes on ice. The remainingpacked red blood cells were stored in the bag at 4° C.

QM (3.2 mg) was mixed with ice cold Adsol (1.6 ml) to make a 2.0 mg/mlsuspension. Aliquots of the QM suspension were added to the cells as setforth in Table 17. The samples were mixed thoroughly by gentle inversionand transferred to Fenwal transfer packs (Baxter/Fenwal, Ill.) forstorage at 4° C.

TABLE 17 Final Concentration Sample of QM (μg/ml) Volume of QM 1 0 0 22.5 37.5 μl 3 5 75 μl 4 10 0.15 ml 5 20 0.30 ml 6 40 0.60 ml

The following measurements of cell function were taken. 1) Potassiumlevels were determined daily for one week and weekly thereafter, usingthe Ciba Corning 614 K⁺/Na⁺ analyzer (commercially available from CibaCorning, Mass.). 2) Adenosine-5′-triphosphate (ATP) and2,3-diphosphoglyceric acid (2,3-DPG) were measured the first day aftertreatment and weekly thereafter. ATP was measured using a Sigma ATP Kit,commercially available from Sigma, St. Louis Mo., following SigmaProcedure No. 366-UV hereby incorporated by reference. 2,3-DPG wasmeasured using the 2,3-DPG Kit, commercially available from Sigma, St.Louis, Mo. 3) IgG binding to the red blood cell surface was measuredafter day 1 and week 1, using the Baxter Unival Anti-Human GlobulinAnti-IgG for Direct Antiglobulin Test and Baxter Coombs Control Cellsfor Quality Control of Anti-Human Globulin Test, commercially availablefrom Baxter Healthcare, Inc., Deerfield, Ill.

The results for R17 inactivation appear in FIG. 7. The results for redblood cell function appear in Tables 18A-18D.

TABLE 18A QM K⁺ K⁺ K⁺ K⁺ K⁺ K⁺ Concentration Day 1 Day 2 Day 7 Day 8 Day9 Day 16 control 7.41 7.41 13.20 12.96 15.32 31.72 2.5 μg/ml  7.42 7.4212.90 13.64 15.86 31.76  5 μg/ml 7.24 7.24 13.28 14.82 15.18 32.96 10μg/ml 7.24 7.24 13.06 15.16 14.68 31.64 20 μg/ml 7.00 7.00 12.90 15.3814.44 31.36 40 μg/ml 6.95 6.95 12.88 12.44 15.44 30.92

TABLE 18B QM ATP (mM) ATP (mM) ATP (mM) Concentration Day 1 Day 9 Day 16control 0.77 0.80 0.75 2.5 μg/ml 0.78 0.80 0.74   5 μg/ml 0.78 0.81 0.73 10 μg/ml 0.76 0.81 0.73  20 μg/ml 0.78 0.80 0.73  40 μg/ml 0.76 0.800.72

TABLE 18C QM 2,3-DPG 2,3-DPG 2,3-DPG Concentration Day 1 Day 9 Day 16control 2.20 0.77 0.89 2.5 μg/ml 2.20 0.88 0.10   5 μg/ml 2.35 0.97 0.28 10 μg/ml 2.06 1.11 0.34  20 μg/ml 2.63 1.43 1.36  40 μg/ml 2.09 1.040.11

TABLE 18D mean median mean median mean median QM FL FL FL FL FL FLConcentration Day 1 Day 1 Day 9 Day 9 Day 16 Day 16 control 4.41 4.14.41 4.1 4.41 4.1 2.5 μg/ml  4.77 4.45 4.77 4.45 4.77 4.45  5 μg/ml 4.794.45 4.79 4.45 4.79 4.45 10 μg/ml 4.96 4.7 4.96 4.7 4.96 4.7 20 μg/ml5.73 5.19 5.73 5.19 5.73 5.19 40 μg/ml 6.31 6.04 6.31 6.04 6.31 6.04

Under conditions of effective R17 inactivation in packed red bloodcells, there are no significant effects on potassium-leakage, ATPcontent or 2,3-DPG content, and only modest effects on IgG binding toRBCs.

EXAMPLE 12

This example evaluates QM to determine whether it is mutagenic in theAmes test, a well known assay for mutagenicity. While mustards areproving to be effective compounds for pathogen inactivation, they arealso considered potential mutagens. This example shows that bloodtreated with QM does not exhibit significant mutagenic action,particularly after an incubation period. Thus, the compounds of thepresent invention have exceptional pathogen inactivation efficiencywhile displaying only minimal mutagenicity.

In this example QM was tested for its mutagenicity using an Ames assay.The mutagenicity was tested under four conditions: QM incubatedovernight in water, QM added to red blood cells and immediately plated;QM added to red blood cells, incubated overnight at 4° C. and thenplated; and QM added to red blood cells, incubated 4 hours at 4° C.,then mixed with Amberlite XAD-16™ and incubated overnight beforeplating.

First, the solubility of QM in red blood cells was determined. 10 mg/mlQM was diluted 10-fold and 100-fold into red blood cells and thesolubility was observed. To obtain a 1.0 mg/ml solution, 20 μl of the 10mg/ml QM solution was combined with 180 μl of the 50% Htc red bloodcells. This stock contained definite particles. Next a 0.3 mg/mlconcentration was tested by combining 20 μl of 3.0 mg/ml QM and 180 μlof 50% Htc red blood cells. There was evidence of precipitating out ofsolution with the 3 mg/ml stock. Finally, 20 μl of the 1.0 mg/ml stockwas mixed with 180 μl of packed red blood cells. The 1.0 mg/ml stockappeared clear. The 3 mg/ml stock was chosen as the highestconcentration, thus 0.3 mg/ml in red blood cells and 30 μg/plate are theupper concentration limits in this experiment.

Preparation of these three test mixtures was as follows. A 10 mg/mlsolution of QM in DMSO was diluted to 1.0 mg/ml (60 μl of QM solutionadded to 0.54 ml DMSO). A 50% Htc red blood cell solution was preparedby spinning down 10 ml of a packed red cell unit at 1600 rpm for 9minutes. Supernatant was removed and the cell pellet was resuspended inan equal volume of Adsol. Htc was then confirmed on a Model F800 Sysmexcell counter (Toa Medical Electronics, Kobe, Japan). Thirteen 0.9 mlaliquots of RBC solution were then placed in test tubes. Four differentstock solutions of QM were prepared because the compound may precipitateout of solution at concentrations as low as 3 mg/ml. Stock solutions atvarying concentrations were prepared by making the following dilutionsof a 1.0 mg/ml solution: 150 μl of a 1.0 mg/ml QM solution+350 μl DMSOto produce a 0.3 mg/ml solution; 40 μl of a 1.0 mg/ml QM solution+360 μlDMSO to produce a 0.1 mg/ml solution; 15 μl of a 1.0 mg/ml QMsolution+485 μl DMSO to produce a 0.03 mg/ml solution. To the firsttube, 100 μl DMSO was added and the tube was placed on a 4° C. shaker(25 rpm, Orbital Shaker, commercially available from VWR Scientific,Greenbelt, Mo.) for overnight incubation. Tubes 2-5 were shakenovernight as well, then 100 μl aliquots of each QM solution was dilutedinto the tubes just before addition to the Ames strains. To tubes 6-9was added 100 μl of each QM solution. The tubes were then incubatedovernight at 4° C. on the shaker. Finally, 100 μl of each QM solutionwas also added to tubes 10-13, which were then incubated on the shakerfor 4 hours. Subsequently, 0.1 g of a polymeric adsorbent material,Amberlite XAD 16™ (commercially available from Sigma, Saint Louis, Mo.),was added to each of tubes 10-13 and the incubation was continuedovernight. The final contents of each tube, and the stock QM solutionsused, are listed in Table 19, below.

TABLE 19 SAMPLE NUMBER CONTENTS QM STOCK SOLUTION 1 RBC + DMSO none 2RBC + 0.003 mg/ml QM 0.03 mg/ml 3 RBC + 0.01 mg/ml QM 0.1 mg/ml 4 RBC +0.03 mg/ml QM 0.3 mg/ml 5 RBC + 0.1 mg/ml QM 1 mg/ml 6 RBC + 0.003 mg/mlQM 0.03 mg/ml 7 RBC + 0.01 mg/ml QM 0.1 mg/ml 8 RBC + 0.03 mg/ml QM 0.3mg/ml 9 RBC + 0.1 mg/ml QM 1 mg/ml 10 RBC + 0.003 mg/ml QM 0.03 mg/ml 11RBC + 0.01 mg/ml QM 0.1 mg/ml 12 RBC + 0.03 mg/ml QM 0.3 mg/ml 13 RBC +0.1 mg/ml QM 1 mg/ml

In a separate experiment, samples of QM in water were prepared asfollows. Sample tubes were labeled and 0.5 ml phosphate buffer was addedto each one. Then various dilutions of a stock solution of QM (1 mg/ml)were added to five of the tubes. For 110 μg/plate−1.4 ml stock solution;for 30 μg/plate−0.42 ml stock+0.98 ml H₂O; for 10 μ/plate−0.14 mlstock+1.26 ml H₂O; for 3 μg/plate−0.042 ml stock+1.358 ml H₂O; for 1μg/plate−0.014 ml stock+1.386 ml H₂O. A control was also prepared usingonly H₂O.

The procedures used for the Salmonella mutagenicity test as described indetail by Maron and Ames were followed exactly. Maron, D. M. and B. N.Ames, Mutation Research 113: 173 (1983). A brief description for eachprocedure is given here. The tester strains TA97a, TA98, TA100, TA102,TA1537 and TA1538 were obtained from Dr. Ames. TA97a, TA98, TA1537 andTA1538 are frame shift tester strains. TA100 and TA102 arebase-substitution tester strains. Upon receipt each strain was culturedunder a variety of conditions to confirm the genotypes specific to thestrains.

The standard Salmonella tester strains used in this study requirehistidine for growth since each tester strain contains a different typeof mutation in the histidine operon. In addition to the histidinemutation, these tester strains contain other mutations, described below,that greatly increase their ability to detect mutagen.

Histidine Dependence: The requirement for histidine was tested bystreaking each strain first on a minimal glucose plate supplemented onlywith biotin and then on a minimal glucose plate supplemented with biotinand histidine. All strains grew only on the histidine/biotinsupplemented plates, confirming a histidine requirement.

rfa Mutation: A mutation which causes partial loss of thelipopolysaccharide. barrier that coats the surface of the bacteria thusincreasing permeability to large molecules was confirmed by exposing astreaked nutrient agar plate coated with the tester strain to crystalviolet. First 100 μL of each culture was added to 2 mL of molten minimaltop agar and poured onto a nutrient agar plate. Then a sterile filterpaper disc saturated with crystal violet was placed at the center ofeach plate. After 16 hours of incubation at 37° C. the plates werescored and a clear zone of no bacterial growth was found around thedisc, confirming the rfa mutation.

uvrB Mutation: Three strains used in this study contain a deficient UVrepair system (TA97a, TA98, TA100, TA1537 and TA1538). This trait wastested for by streaking the strains on a nutrient agar plate, coveringhalf of the plate, and irradiating the exposed side of the plate withgermicidal lamps. After incubation growth was only seen on the side ofthe plate shielded from UV irradiation.

R-factor: The tester strains (TA97a, TA98, TA100, and TA102) contain thepKM101 plasmid that increases their sensitivity to mutagens. The plasmidalso confers resistance to ampicillin to the bacteria. This wasconfirmed by growing the strains in the presence of ampicillin.

pAQ1: Strain TA102 also contains the pAQ1 plasmid that further enhancesits sensitivity to mutagens. This plasmid also codes for tetracyclineresistance. To test for the presence of this plasmid TA102 was streakedon a minimal glucose plate containing histidine, biotin, andtetracycline. The plate was incubated for 16 hours at 37° C. The strainshowed normal growth indicating the presence of the pAQ1 plasmid.

The same cultures used for the genotype testing were again cultured andaliquots were frozen under controlled conditions. The cultures wereagain tested for genotype to confirm the fidelity of the genotype uponmanipulation in preparing the frozen permanents.

The first tests done with the strains were to determine the range ofspontaneous reversion for each of the strains. With each mutagenicityexperiment the spontaneous reversion of the tester strains to histidineindependence was measured and expressed as the number of spontaneousrevertants per plate. This served as the background controls. A positivemutagenesis control was included for each tester strain by using adiagnostic mutagen suitable for that strain (2-aminofluorene at 5mg/plate for TA98; sodium azide at 1.5 mg/plate for TA100;9-aminoacridine for TA 1537).

For all experiments, the pre-incubation procedure was used. In thisprocedure one vial of each tester strain was thawed and tubes wereprepared for each strain, containing 20 μL of the culture and 6 mL ofOxoid Nutrient Broth #2. This solution was allowed to shake for 10 hoursat 37° C. In the pre-incubation procedure, for each tester strain usedto evaluate the test solution, 0.1 mL of the overnight culture was addedto each of 13 sterile test tubes. To each of the tubes, 0.1 mL of thetest solution from tubes 1-13 was added. This was also performed on thesamples containing QM in water only. Then 0.5 mL of 0.2 M sodiumphosphate buffer, pH 7.4 was added. The 0.7 mL mixture was vortexed andthen pre-incubated while shaking for 20 minutes at 37° C. After shaking,2 mL of molten top agar supplemented with histidine and biotin wereadded to the 0.7 mL mixture and immediately poured onto a minimalglucose agar plate (volume of base agar was 20 mL). The top agar wasallowed 30 minutes to solidify and then the plates were inverted andincubated for 44 hours at 37° C. After incubation, the number ofrevertant colonies on each plate was counted.

The results appear in FIG. 8. Although the QM registered a positiveresponse in the Ames test without incubation in red blood cells and inwater, an overnight incubation in red blood cells significantly reducedthe level of revertants, as did an incubation with adsorbent material. Aparallel experiment was performed using an activated charcoal adsorbentmaterial, Hemosorba (commercially available from Asahi Medical Corp.,Tokyo, Japan.) The results, which are not shown, were similar to theresults using Amberlite XAD 16™.

EXAMPLE 13

As discussed above, a method of decontaminating clinical samples wouldbe most useful if while it decontaminated samples, it did notsignificantly effect the results of the clinical tests themselves. Thisexample compares results of a common blood chemistry panel for samplestreated by methods of the present invention to untreated samples.

Solutions of QM and ICR-170 (2 mg/ml) were prepared in saline. The QMwas almost completely dissolved, and ICR-170 remained a suspension.Next, human whole blood was drawn and 10 ml aliquots were placed ineight tubes. QM or ICR-170 was added to six of the tubes in aliquots of100, 200, or 400 μl, to reach final concentrations of the compounds ofeither 20, 40, or 80 μg/ml. Saline was added to the remaining two tubes,in aliquots of 100 or 400 μl, to prepare control samples. The sampleswere then allowed to clot for 30 minutes, followed by 20 minutes on acentrifuge at 1000 rpm. The separated serum was then transferred tolabeled plastic tubes and tested in a panel of 24 common clinicalchemistry tests.

The results appear below, in Table 20. Neither QM nor ICR-170 had asignificant effect on the results of any of the panel of 24 clinicalchemistry tests. Lactate dehydrogenase and GGT exhibited a small drop inthe sample containing the highest concentration of ICR-170. Clearly, themethods of the present invention do not interfere significantly withclinical testing of blood samples.

TABLE 20 100 μl 400 μl 20 μg/ml 40 μg/ml 60 μg/ml 20 μg/ml 40 μg/ml 60μg/ml TEST saline saline QM QM QM ICR170 ICR170 ICR170 Glucose 95 92 9393 90 94 92 90 BUN 17 17 17 17 17 17 17 17 Creatinine 1.1 1.1 1.1 1.21.1 1 1.1 1.1 Bun/Creat. ratio 15 15 15 14 15 17 15 15 Sodium 143 143138 144 146 143 144 144 Potassium 4 3.9 4 3.9 3.9 4 4.1 4.1 Chloride 104106 104 106 107 103 104 103 Magnesium 1.7 1.6 1.6 1.6 1.6 1.7 1.6 1.5Calcium 9.3 8.8 9.3 8.8 9 9.4 9.2 9.1 Phosphorous 4.3 3.9 4.1 4.1 4.14.3 4.3 4.1 inorganic protein, total 7.3 7.1 7.4 7.3 7.1 7.3 7.3 7.1albumin 4.6 4.5 4.6 4.6 4.4 4.6 4.6 4.4 globulin, total 2.7 2.6 2.8 2.72.7 2.7 2.7 2.7 A/G ratio 1.7 1.7 1.6 1.7 1.6 1.7 1.7 1.6 billirubin 0.60.6 0.6 0.6 0.6 0.6 0.6 0.6 alk 55 50 48 50 51 54 51 47 LDH 136 134 121125 138 129 138 151 GGT 23 21 22 22 22 23 20 16 AST 17 16 18 16 16 16 1716 ALT 20 19 17 19 18 19 19 19 Uric Acid 5.6 5.3 5.5 5.4 5.4 5.7 5.6 5.5Iron 119 107 117 113 115 119 122 120 Triglycerides 164 152 159 158 158161 150 156 Cholesterol 234 221 227 226 223 231 231 223

EXAMPLE 14

This example describes the inactivation of bacterial pathogens ofbiological compositions using methods of the present invention. Thefollowing experiment was performed to support that the methods of thepresent invention can be used to inactivate bacterial pathogens. In thisexample, the decontamination methods of the present invention wereapplied to inactivate Staphylococcus epidermis.

An overnight culture of the organism was made by inoculating 3 ml of LBbroth from a motility stab. This was maintained at 35° C. and 1.0 ml ofit was used to inoculate 9 ml of LB broth in a 15 ml conical tube. Asample (1 ml) was taken for an OD₆₀₀ reading. To a tube, 5 ml of LBbroth was added. Then 50 μl of 10⁸ cfu/ml S. epidermis was added.Aliquots of the sample (1 ml each) were placed in 5 tubes. These weretreated with either 0, 3, 10, 30, or 100 μg/ml of QM. A 2 mg/ml stock ofQM in ddH₂O was added in the following amounts to produce the desiredconcentrations: 0 μl, 1.5 μl, 5 μl, 15 μl, and 50 μl. The samples wereincubated on ice for three hours.

After incubation, bacteria was quantified by plating 0.1 ml of serial10-fold dilutions in LB broth onto 100 mm petri dishes containing agar.After 24 hr. incubation at 35° C., colonies were counted and bacterialconcentration was calculated on a per ml basis. The results, whichappear in FIG. 9, show that QM at >10 μg/ml inactivates S. epidermis tothe level of detection of this assay.

EXAMPLE 15

If a decontaminated blood product is to have value as an in vivotherapy, the blood product must retain some efficacy after thedecontamination process. One way to confirm the efficacy of a particularsample of red blood cells is to ensure that they are not cleared by therecipient's body significantly sooner than normal red blood cells whentransfused into a mammal. In this example, packed red blood cells aretreated with compounds having a nucleic acid binding ligand and amustard group, according to the methods of the present invention,transfused into mice, and tracked for post transfusion survival.

Blood was drawn from 8 Balb/c mice using an anticoagulant (containingcitrate, ethylene diamine tetraacetic acid, prostaglandin El andtheophyllin) for a total of 12 ml (8 ml whole blood and 4 mlanticoagulant). An equivalent volume of Adsol was added and the samplewas centrifuged at 2000 rpm for 5 minutes. The supernatant was removedand saved as “washed solution”. The red blood cell pellet wasresuspended in Adsol to make a 50% Htc solution. Three 1.5 ml aliquotswere transferred to 14 ml round bottom polypropylene tubes.

Next, 1 mg/ml solutions of two compounds, QM and ICR-170, were preparedin saline. The compounds were added to the three polypropylene tubes asfollows: tube 1 received no treatment (120 μl of saline was added); tube2 received 120 μl of the 1 mg/ml solution of QM for a finalconcentration of 80 μg/ml; tube 3 120 μl of the 1 mg/ml solution ofICR-170, for a final concentration of 80 μg/ml.

The samples were then incubated for 2 hours at 4° C. Cells were washedthree times, each time by adding 6 ml Adsol to each tube and spinningthe samples at 1800 rpm for 5 minutes. After the final wash, the pelletwas resuspended in Adsol buffer to a concentration of 4×10⁶ cells/μl. 50μl of each sample was removed for an unstained control.

The remaining cells were then stained with PKH26 dye. From a 1mM PKH26stock, 120 μl was removed and diluted with 8 ml diluent A to create aworking solution of 15.7 μM PKH26. This solution was stored in the darkuntil use. To each 2 ml of cells, 2 ml of PKH26 dye was added. Thesamples were mixed gently and incubated for 5 minutes at roomtemperature in the dark. The cells were remixed after 2.5 minutes. Afteranother 5 minute incubation, 2 volumes of the reserved “washed solution”was added to stop the staining reaction. The cells were centrifuged at1800 rpm for 5 minutes to pellet and the supernatant was removed. Thecells were then washed 3 times with Adsol buffer, as before. After thefinal wash, the sample volume was restored to 1 ml with Adsol. Analiquot of each sample was removed at this point for a positive stainedcontrol sample and counted on the Sysmex machine.

Swiss Webster mice were transfused with 0.2 ml of the labeled cells fromeach of samples 1-3 via tail vein injection. The mice were then weighedto calculate the blood volume. Blood volume is calculated as the animalweight in gm×0.06. Then, blood was drawn from the mice by retroorbitalvenipuncture using heparin-EDTA coated capillary tubes at 1 hour, 24hours, 2 more times during the first week, and one time weekly for 3weeks. The eye bleeding samples were drained into isotonic solutionbefore analysis.

Samples were analyzed on a FACScan™ at the FL2 (red fluorescent channel)with gating on the red cell population using forward and side scatterlinear mode gates. The proportion of labeled cells in 100,000 total redcell gated events was determined.

TABLE 21 SAMPLE RECOVERY LOSS PER DAY Untreated 90.3 ± 3.2 2.66% QM 80.8± 4.7 2.77% ICR-170 86.0 ± 6.6 2.71%

The results appear in Table 21. According to the results, regardless oftreatment, treated cells survived in vivo as well as untreated controlcells.

Some hemolysis of RBC was detected after labeling with PKH26. Thus,recoveries may be effected by the labeling technique. An alternativelabeling technique was also used, as described. An activated biotinester was injected via the tail vein of the Balb/c mice in order tolabel the red cells in vivo (each mouse received either a “low dosetreatment” −0.1 mg injection on two successive days, or a “high dosetreatment” −0.3 mg of biotin on three successive days). After treatment,low and high dose treatment cells with fluorescently tagged streptavidinwere clearly distinguishable as detected by FACScan analysis. Low andhigh dose treatment.,cells were independently treated with 80 μg/ml ofQM or ICR-170 as described above. After treatment, cells were washed,mixed with untreated cells that had been differentially labeled. Thenthree mice were transfused: one received untreated low and untreatedhigh dose RBC, one received QM treated low and untreated high dose RBC,and one received ICR-170 treated low and untreated high RBC. Bleedingwas performed as described above. The results appear in Table 22, below.Clearly, recovery of the treated cells is very similar to the untreatedcells.

TABLE 22 % RECOVERY, LOW % RECOVERY, HIGH untreated 96.2 93.5 QM 94.691.2 ICR-170 93.1 90.4

EXAMPLE 16

This example describes the synthesis of a compound of the presentinvention, 5-[N,N-bis(2-chloroethyl)amino]methyl-8-methoxypsoralenhydrochloride (referred to throughout the text as “Compound 1”).

Step 1: The synthesis of 5-Bromomethyl-8-methoxypsoralen.

To a solution of 2.69 g(12.3 mmol) of 8-methoxypsoralen (commerciallyavailable from Aldrich, Milwaukee, Wis.) in 135 mL of glacial aceticacid was added 11 mL of bromomethyl methyl ether. The solution wasswirled, then left for three days at room temperature during which awhite solid precipitated. The mixture was cooled in an ice bath andfiltered. To the filtrate was added an additional 2.75 g (12.7 mmol) of8-methoxypsoralen and 5 mL of BrCH₂OCH₃. After again sitting for threedays, product isolation was repeated. The filter cakes were washed withcold glacial acetic acid, air dried and finally vacuum dried to give atotal yield of 6.3 g (81%) of 5-bromomethyl-8-methoxypsoralen as a paleyellow solid. NMR (CDCl3): 4.33 (S, 3H), 4.88 (s, 2H), 6.52 (d, J=10 Hz,1H), 6.95 (d, J=2 Hz, 1H), 7.77 (d, J=2 Hz, 1H), 8.11 (d, J=10 Hz, 1H).

Step 2: The synthesis of5-[N,N-Bis-(2-hydroxyethyl)amino]methyl-8-methoxypsoralen.

5-Bromomethyl-8-methoxypsoralen (0.50 g, 1.6 mmol, from Step 1, above)and diethanolamine (2.56 mL, 27 mmol) were combined in 23 mL of absoluteethanol and refluxed for 10 hours. The solution was concentrated, thenCHCl₃ (65 mL) was added to the residue. The organic layer was washedwith 30, 30 and 10 mL of water sequentially and the combined aqueoussolutions were back extracted with CHCl₃. The combined organic solutionswere then extracted three times, each time with 7 mL of 1.2 N HCl. Thecombined acid solution was taken to pH5-6 with 10% aqueous NaOH and theresultant turbid solution was washed 4 times, each time with 20 mL ofCHCl₃. This last organic solution was rinsed with 2×20 mL of brine,dried (Na₂SO₄) and concentrated to give 0.43 g (79%) of the aminediol,5-[N,N-bis-(2-hydroxyethyl)amino]methyl-8-methoxypsoralen, melting point121-122° C.; NMR (CDCl3): 2.71 (t, J=5 Hz, 4H), 3.61 (t, J=5 Hz, 4H),4.09 (s, 2H), 4.29 (s, 3H), 6.41 (d, J=10 Hz, 1H), 7.00 (d, J=2 Hz, 1H),7.70 (d, J=2 Hz, 1H), 8.38 (d, J=10 Hz, 1H).

Step 3: 5-[N,N-Bis(2-chloroethyl)amino]methyl-8-methoxypsoralenhydrochloride.

5-[N,N-Bis(2-hydroxyethyl)amino]methyl-8-methoxypsoralen (0.030 g, 0.090mmol) was dissolved in 1 ml thionyl chloride. It was covered with aserum cap with a small needle vent and allowed to stir for 3 days. Thereaction mixture was stripped and the crude solid was recrystallized inisopropanol to give5-[N,N-bis(2-chloroethyl)amino]methyl-8-methoxypsoralen hydrochloride(0.012 g, 32.4%) as an off-white solid, mp 158-162° C. ¹HNMR (CD₃OD):3.40 (t, J=6 Hz, 4H), 3.86 (t, J=6 Hz, 4H), 4.33 (s, 3H), 4.70 (s, 2H),6.52 (d, J=10 Hz, 1H), 7.28 (d, J=2 Hz, 1H), 8.03 (d, J=2 Hz, 1H), 8.48(d, J=10 Hz, 1H). The chemical shift appeared to be sensitive to traceacid present.

A portion of the above salt was partitioned between methylene chlorideand aqueous NaHCO₃. The organic layer was again washed with aqueousbicarbonate, dried with brine, then dried with anhydrous Na₂SO₄ andevaporated to give the neutral amine; mass spectrum (EL, m/e): 371 (3),369 (4), 230 (16), 229 (100), 214 (5), 201 (5), 186 (10) (obtained on aShimadzu QP5000 GC/MS, with Rtx-5,15 m column, commercially availablefrom Shimadzu Corporation, Kyoto, Japan).

EXAMPLE 17

This example describes a contemplated embodiment wherein red blood cellsare treated by a method of the present invention. The standard bloodproduct separation approach used presently in blood banks is as follows:three bags are integrated by flexible tubing to create a blood transferset (e.g., commercially available from Baxter, Deerfield, Ill.). Afterblood is drawn into the first bag, the entire set is processed bycentrifugation (e.g., Sorvall™ swing bucket centrifuge, Dupont),resulting in packed red cells and platelet rich plasma in the first bag.The plasma is expressed off of the first bag (e.g., using a Fenwall™device for plasma expression), through the tubing and into the secondbag. The first bag, containing packed red cells, is then detached.

In one embodiment of the decontamination approach of the presentinvention applied specifically to red blood cells, a compound having anucleic acid binding ligand and a mustard group is introduced to the redblood cells (e.g. the compound may be present in the first bag beforeblood is drawn, or transferred to the first bag after centrifugation)and incubated. After incubation, the compound may be removed using anadsorbent material (e.g., a commercially available material, such asactivated charcoal or an Amberlite resin). The adsorbent may beintroduced directly into the bag containing the red blood cells, or thered blood cells may be passed through a scrub device which contains theadsorbent. The incubation, scrub, and any subsequent storage, may takeplace in a commercially available storage bag.

From the above, it should be evident that the present invention providesmethods of decontamination of blood preparations intended for storageand in vivo use.

It is to be understood that the present invention is not to be limitedto the exact details of operation or exact compounds, compositions,methods, or procedures shown and described, as modifications andequivalents will be apparent to one skilled in the art. All patentsdescribed are hereby incorporated by reference.

We claim:
 1. An in vitro method of inactivating a pathogen in mammalianblood or a mammalian blood product comprising: (a) contacting in vitromammalian blood or a mammalian blood product, with a pathogeninactivating amount of a first compound that has an intercalator nucleicacid binding ligand and a moiety selected from the group consisting of amustard group, a mustard group equivalent, and a mustard groupintermediate attached thereto, which when incubated with the blood orblood product results in the inactivation of at least 1 log of pathogenpresent in the blood or blood product, if any, (b) and recovering thepathogen inactivated blood or blood product, wherein the recovered bloodor blood product has substantially the same biological activity suchthat it is suitable for therapeutic use in a mammal, wherein the firstcompound has a greater inactivation efficiency against R17 than a secondcompound containing a mustard group, mustard group equivalent, ormustard group intermediate that lacks the intercalator nucleic acidbinding ligand.
 2. The method according to claim 1 wherein the pathogenis a RNA-containing pathogen.
 3. The method according to claim 2 whereinthe pathogen is HIV.
 4. The method according to claim 1 wherein thepathogen is a DNA-containing pathogen.
 5. The method according to claim4 wherein the pathogen is a hepatitis virus.
 6. The method according toclaim 1, wherein the contacting in vitro is for a time between 1 minuteand 48 hours.
 7. The method according to claim 1, wherein the firstcompound is present at a concentration of 320 micromolar or less.
 8. Themethod according to claim 1, further comprising: c) transfusing therecovered product into a mammal.
 9. The method according to claim 1,further comprising: c) removing the first compound from the blood orblood product with an adsorbent material.
 10. The method according toclaim 9, further comprising: d) transfusing the recovered product into amammal.
 11. The method according to claim 1, wherein the intercalatornucleic binding ligand is an acridine.
 12. The method according to claim11, wherein the acridine is a 9-aminoacridine.
 13. The method accordingto claim 12, wherein the first compound isN-2-chloroethyl)-N-ethyl-N′-(6-chloro-2-methoxy-9-acridinyl)-1,3-propanediaminedihydrochloride.
 14. The method according to claim 1, wherein theintercalator nucleic acid binding ligand is a psoralen.
 15. The methodaccording to claim 1, wherein the moiety is a mustard group equivalentand the mustard group equivalent is an epoxide.
 16. The method accordingto claim 1, wherein the moiety is a mustard group equivalent and themustard group equivalent is an aziridine.
 17. The method according toclaim 1, wherein the blood product comprises red blood cells.
 18. Themethod according to claim 1, wherein the treatment is of whole blood.19. The method according to claim 17 wherein the mammal is a human. 20.The method according to claim 18 wherein the mammal is a human.